8-fluorophthalazin-1(2H)-one compounds

ABSTRACT

8-Fluorophthalazin-1(2h)-one compounds of Formula I where one or two of X 1 , X 2 , and X 3  are N, are provided, including stereoisomers, tautomers, and pharmaceutically acceptable salts thereof, useful for inhibiting Btk kinase, and for treating immune disorders such as inflammation mediated by Btk kinase. Methods of using compounds of Formula I for in vitro, in situ, and in vivo diagnosis, and treatment of such disorders in mammalian cells, or associated pathological conditions, are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/667,126, filed 2Nov. 2012, and also claims the benefit of priority under 35 USC §119(e)of U.S. Provisional Application Ser. No. 61/555,398 filed on 3 Nov.2011, which are incorporated by reference in entirety.

FIELD OF THE INVENTION

The invention relates generally to compounds for treating disordersmediated by Bruton's Tyrosine Kinase (Btk) including inflammation,immunological, and cancer, and more specifically to compounds whichinhibit Btk activity. The invention also relates to methods of using thecompounds for in vitro, in situ, and in vivo diagnosis or treatment ofmammalian cells, or associated pathological conditions.

BACKGROUND OF THE INVENTION

Protein kinases, the largest family of human enzymes, encompass wellover 500 proteins. Bruton's Tyrosine Kinase (Btk) is a member of the Tecfamily of tyrosine kinases, and is a regulator of early B-celldevelopment as well as mature B-cell activation, signaling, andsurvival.

B-cell signaling through the B-cell receptor (BCR) can lead to a widerange of biological outputs, which in turn depend on the developmentalstage of the B-cell. The magnitude and duration of BCR signals must beprecisely regulated. Aberrant BCR-mediated signaling can causedisregulated B-cell activation and/or the formation of pathogenicauto-antibodies leading to multiple autoimmune and/or inflammatorydiseases. Mutation of Btk in humans results in X-linkedagammaglobulinaemia (XLA). This disease is associated with the impairedmaturation of B-cells, diminished immunoglobulin production, compromisedT-cell-independent immune responses and marked attenuation of thesustained calcium sign upon BCR stimulation. Evidence for the role ofBtk in allergic disorders and/or autoimmune disease and/or inflammatorydisease has been established in Btk-deficient mouse models. For example,in standard murine preclinical models of systemic lupus erythematosus(SLE), Btk deficiency has been shown to result in a marked ameliorationof disease progression. Moreover, Btk deficient mice can also beresistant to developing collagen-induced arthritis and can be lesssusceptible to Staphylococcus-induced arthritis. A large body ofevidence supports the role of B-cells and the humoral immune system inthe pathogenesis of autoimmune and/or inflammatory diseases.Protein-based therapeutics (such as Rituxan) developed to depleteB-cells, represent an approach to the treatment of a number ofautoimmune and/or inflammatory diseases. Because of Btk's role in B-cellactivation, inhibitors of Btk can be useful as inhibitors of B-cellmediated pathogenic activity (such as autoantibody production). Btk isalso expressed in osteoclasts, mast cells and monocytes and has beenshown to be important for the function of these cells. For example, Btkdeficiency in mice is associated with impaired IgE-mediated mast cellactivation (marked diminution of TNF-alpha and other inflammatorycytokine release), and Btk deficiency in humans is associated withgreatly reduced TNF-alpha production by activated monocytes.

Thus, inhibition of Btk activity can be useful for the treatment ofallergic disorders and/or autoimmune and/or inflammatory diseases suchas: SLE, rheumatoid arthritis, multiple vasculitides, idiopathicthrombocytopenic purpura (ITP), myasthenia gravis, allergic rhinitis,and asthma (Di Paolo et al (2011) Nature Chem. Biol. 7(1):41-50; Liu etal (2011) Jour. of Pharm. and Exper. Ther. 338(1):154-163). In addition,Btk has been reported to play a role in apoptosis; thus, inhibition ofBtk activity can be useful for cancer, as well as the treatment ofB-cell lymphoma, leukemia, and other hematological malignancies.Moreover, given the role of Btk in osteoclast function, the inhibitionof Btk activity can be useful for the treatment of bone disorders suchas osteoporosis. Specific Btk inhibitors have been reported (Liu (2011)Drug Metab. and Disposition 39(10):1840-1849; U.S. Pat. No. 7,884,108,WO 2010/056875; U.S. Pat. No. 7,405,295; U.S. Pat. No. 7,393,848; WO2006/053121; U.S. Pat. No. 7,947,835; US 2008/0139557; U.S. Pat. No.7,838,523; US 2008/0125417; US 2011/0118233; PCT/US2011/050034“PYRIDINONES/PYRAZINONES, METHOD OF MAKING, AND METHOD OF USE THEREOF”,filed 31 Aug. 2011; PCT/US2011/050013 “PYRIDAZINONES, METHOD OF MAKING,AND METHOD OF USE THEREOF”, filed 31 Aug. 2011; U.S. Ser. No. 13/102,720“PYRIDONE AND AZA-PYRIDONE COMPOUNDS AND METHODS OF USE”, filed 6 May2011).

SUMMARY OF THE INVENTION

The invention relates generally to Formula I and II compounds,8-fluorophthalazin-1(2h)-one compounds with Bruton's Tyrosine Kinase(Btk) modulating activity, having the structures:

including stereoisomers, tautomers, or pharmaceutically acceptable saltsthereof. The various substituents are defined herein below.

One aspect of the invention is a pharmaceutical composition comprised ofa Formula I or II compound, and a pharmaceutically acceptable carrier,glidant, diluent, or excipient. The pharmaceutical composition mayfurther comprise a second therapeutic agent.

Another aspect of the invention is a process for making a pharmaceuticalcomposition which comprises combining a Formula I or II compound with apharmaceutically acceptable carrier.

The invention includes a method of treating a disease or disorder whichmethod comprises administering a therapeutically effective amount of aFormula I or II compound to a patient with a disease or disorderselected from immune disorders, cancer, cardiovascular disease, viralinfection, inflammation, metabolism/endocrine function disorders andneurological disorders, and mediated by Bruton's tyrosine kinase.

The invention includes a kit for treating a condition mediated byBruton's tyrosine kinase, comprising: a) a first pharmaceuticalcomposition comprising a Formula I or II compound; and b) instructionsfor use.

The invention includes a Formula I or II compound for use as amedicament, and for use in treating a disease or disorder selected fromimmune disorders, cancer, cardiovascular disease, viral infection,inflammation, metabolism/endocrine function disorders and neurologicaldisorders, and mediated by Bruton's tyrosine kinase.

The invention includes use of a Formula I or II compound in themanufacture of a medicament for the treatment of immune disorders,cancer, cardiovascular disease, viral infection, inflammation,metabolism/endocrine function disorders and neurological disorders, andwhere the medicament mediates Bruton's tyrosine kinase.

The invention includes methods of making a Formula I or II compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the preparation of6-tert-Butyl-8-fluoro-2-(2-(hydroxymethyl)-3-(1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)phthalazin-1(2H)-one101 starting with 4-tert-Butylbenzoyl Chloride 101a

FIG. 2 shows the preparation of6-tert-Butyl-8-fluoro-2-(3-(hydroxymethyl)-4-(1-methyl-5-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-2-yl)phthalazin-1(2H)-one102 starting with 2,6-Dibromo-4-fluorobenzaldehyde 102a

FIG. 3 shows the preparation of6-tert-butyl-8-fluoro-2-(3-(hydroxymethyl)-4-(1-methyl-5-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-2-yl)-phthalazin-1(2H)-one103 starting with 2-Bromo-4-chloronicotinaldehyde 103a

FIG. 4 shows the preparation of(S)-6-tert-butyl-8-fluoro-2-(3-(hydroxymethyl)-4-(1-methyl-5-(3-methyl-5-(4-(oxetan-3-yl)piperazine-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-2-yl)phthalazin-1(2H)-one105 starting with (3S)-tert-Butyl3-Methyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 105a

FIG. 5 shows the preparation ofR-6-tert-Butyl-8-fluoro-2-(3-(hydroxymethyl)-4-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazine-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-2-yl)phthalazin-1(2H)-one106 starting with1-Methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazine-1-yl)pyridin-2-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one106a

FIG. 6 shows the preparation of(S)-6-tert-butyl-8-fluoro-2-(4-(hydroxymethyl)-5-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)phthalazin-1(2H)-one109 starting with3-Bromo-5-(6-tert-butyl-8-fluoro-1-oxo-1,2-dihydrophthalazin-2-yl)pyridine-4-carbaldehyde109a

FIG. 7 shows the preparation of6-tert-Butyl-2-(4-(5-(5-((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-3-(hydroxymethyl)pyridin-2-yl)-8-fluorophthalazin-1(2H)-one110 starting with2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(5-((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde110a

FIG. 8 shows the preparation of(S)-6-tert-Butyl-2-(4-(5-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-3-(hydroxymethyl)pyridin-2-yl)-8-fluorophthalazin-1(2H)-one112 starting with(S)-2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde112a

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents which may be included within the scope ofthe present invention as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention. The present invention is in no way limited to the methods andmaterials described. In the event that one or more of the incorporatedliterature, patents, and similar materials differs from or contradictsthis application, including but not limited to defined terms, termusage, described techniques, or the like, this application controls.Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. The nomenclature used in this Application is based on IUPACsystematic nomenclature, unless indicated otherwise.

Definitions

When indicating the number of substituents, the term “one or more”refers to the range from one substituent to the highest possible numberof substitution, i.e. replacement of one hydrogen up to replacement ofall hydrogens by substituents. The term “substituent” denotes an atom ora group of atoms replacing a hydrogen atom on the parent molecule. Theterm “substituted” denotes that a specified group bears one or moresubstituents. Where any group may carry multiple substituents and avariety of possible substituents is provided, the substituents areindependently selected and need not to be the same. The term“unsubstituted” means that the specified group bears no substituents.The term “optionally substituted” means that the specified group isunsubstituted or substituted by one or more substituents, independentlychosen from the group of possible substituents. When indicating thenumber of substituents, the term “one or more” means from onesubstituent to the highest possible number of substitution, i.e.replacement of one hydrogen up to replacement of all hydrogens bysubstituents.

The term “alkyl” as used herein refers to a saturated linear orbranched-chain monovalent hydrocarbon radical of one to twelve carbonatoms (C₁-C₁₂), wherein the alkyl radical may be optionally substitutedindependently with one or more substituents described below. In anotherembodiment, an alkyl radical is one to eight carbon atoms (C₁-C₈), orone to six carbon atoms (C₁-C₆). Examples of alkyl groups include, butare not limited to, methyl (Me, —CH₃), ethyl (Et, —CH₂CH₃), i-propyl(n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr, i-propyl, —CH(CH₃)₂),1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃), 2-methyl-i-propyl (i-Bu,i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, —CH(CH₃)CH₂CH₃),2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl (n-pentyl,—CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, 1-heptyl, 1-octyl, and the like.

The term “alkylene” as used herein refers to a saturated linear orbranched-chain divalent hydrocarbon radical of one to twelve carbonatoms (C₁-C₁₂), wherein the alkylene radical may be optionallysubstituted independently with one or more substituents described below.In another embodiment, an alkylene radical is one to eight carbon atoms(C₁-C₈), or one to six carbon atoms (C₁-C₆). Examples of alkylene groupsinclude, but are not limited to, methylene (—CH₂—), ethylene (—CH₂CH₂—),propylene (—CH₂CH₂CH₂—), and the like.

The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp² double bond,wherein the alkenyl radical may be optionally substituted independentlywith one or more substituents described herein, and includes radicalshaving “cis” and “trans” orientations, or alternatively, “E” and “Z”orientations. Examples include, but are not limited to, ethylenyl orvinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), and the like.

The term “alkenylene” refers to linear or branched-chain divalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp² double bond,wherein the alkenylene radical may be optionally substitutedindependently with one or more substituents described herein, andincludes radicals having “cis” and “trans” orientations, oralternatively, “E” and “Z” orientations. Examples include, but are notlimited to, ethylenylene or vinylene (—CH═CH—), allyl (—CH₂CH═CH—), andthe like.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical of two to eight carbon atoms (C₂-C₈) with at least one site ofunsaturation, i.e., a carbon-carbon, sp triple bond, wherein the alkynylradical may be optionally substituted independently with one or moresubstituents described herein. Examples include, but are not limited to,ethynyl (—C≡CH), propynyl (propargyl, —CH₂C≡CH), and the like.

The term “alkynylene” refers to a linear or branched divalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp triple bond, whereinthe alkynylene radical may be optionally substituted independently withone or more substituents described herein. Examples include, but are notlimited to, ethynylene (—C≡C—), propynylene (propargylene, —CH₂C≡C—),and the like.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms (C₃-C₁₂) as a monocyclicring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycleshaving 7 to 12 atoms can be arranged, for example, as a bicyclo [4,5],[5,5], [5,6] or [6,6] system, and bicyclic carbocycles having 9 or 10ring atoms can be arranged as a bicyclo [5,6] or [6,6] system, or asbridged systems such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Spiro moieties are also included within the scopeof this definition. Examples of monocyclic carbocycles include, but arenot limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like. Carbocyclyl groups areoptionally substituted independently with one or more substituentsdescribed herein.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of one hydrogen atom from a singlecarbon atom of a parent aromatic ring system. Some aryl groups arerepresented in the exemplary structures as “Ar”. Aryl includes bicyclicradicals comprising an aromatic ring fused to a saturated, partiallyunsaturated ring, or aromatic carbocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, biphenyl, indenyl,indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and thelike. Aryl groups are optionally substituted independently with one ormore substituents described herein.

“Arylene” means a divalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of two hydrogen atom from a twocarbon atoms of a parent aromatic ring system. Some arylene groups arerepresented in the exemplary structures as “Ar”. Arylene includesbicyclic radicals comprising an aromatic ring fused to a saturated,partially unsaturated ring, or aromatic carbocyclic ring. Typicalarylene groups include, but are not limited to, radicals derived frombenzene (phenylene), substituted benzenes, naphthalene, anthracene,biphenylene, indenylene, indanylene, 1,2-dihydronaphthalene,1,2,3,4-tetrahydronaphthyl, and the like. Arylene groups are optionallysubstituted with one or more substituents described herein.

The terms “heterocycle,” “heterocyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to about 20 ring atoms in which atleast one ring atom is a heteroatom selected from nitrogen, oxygen,phosphorus and sulfur, the remaining ring atoms being C, where one ormore ring atoms is optionally substituted independently with one or moresubstituents described below. A heterocycle may be a monocycle having 3to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selectedfrom N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), forexample: a bicyclo[4,5], [5,5], [5,6], or [6,6] system. Heterocycles aredescribed in Paquette, Leo A.; “Principles of Modern HeterocyclicChemistry” (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3,4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series ofMonographs” (John Wiley & Sons, New York, 1950 to present), inparticular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960)82:5566. “Heterocyclyl” also includes radicals where heterocycleradicals are fused with a saturated, partially unsaturated ring, oraromatic carbocyclic or heterocyclic ring. Examples of heterocyclicrings include, but are not limited to, morpholin-4-yl, piperidin-1-yl,piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one,pyrrolidin-1-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl,azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl,[1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl,dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolizinyl and N-pyridyl ureas. Spiro moieties are also includedwithin the scope of this definition. Examples of a heterocyclic groupwherein 2 ring atoms are substituted with oxo (═O) moieties arepyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocycle groupsherein are optionally substituted independently with one or moresubstituents described herein.

The term “heteroaryl” refers to a monovalent aromatic radical of 5-, 6-,or 7-membered rings, and includes fused ring systems (at least one ofwhich is aromatic) of 5-20 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl(including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl,oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl,isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl,benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl,pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl,triazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl,benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl,quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups areoptionally substituted independently with one or more substituentsdescribed herein.

The heterocycle or heteroaryl groups may be carbon (carbon-linked), ornitrogen (nitrogen-linked) bonded where such is possible. By way ofexample and not limitation, carbon bonded heterocycles or heteroarylsare bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5,or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or β-carboline.

The terms “treat” and “treatment” refer to therapeutic treatment,wherein the object is to slow down (lessen) an undesired physiologicalchange or disorder, such as the development or spread of arthritis orcancer. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation of symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Those in need of treatment include those with the conditionor disorder.

The phrase “therapeutically effective amount” means an amount of acompound of the present invention that (i) treats the particulardisease, condition, or disorder, (ii) attenuates, ameliorates, oreliminates one or more symptoms of the particular disease, condition, ordisorder, or (iii) prevents or delays the onset of one or more symptomsof the particular disease, condition, or disorder described herein. Inthe case of cancer, the therapeutically effective amount of the drug mayreduce the number of cancer cells; reduce the tumor size; inhibit (i.e.,slow to some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy can be measured, for example, by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR).

“Inflammatory disorder” as used herein can refer to any disease,disorder, or syndrome in which an excessive or unregulated inflammatoryresponse leads to excessive inflammatory symptoms, host tissue damage,or loss of tissue function. “Inflammatory disorder” also refers to apathological state mediated by influx of leukocytes and/or neutrophilchemotaxis.

“Inflammation” as used herein refers to a localized, protective responseelicited by injury or destruction of tissues, which serves to destroy,dilute, or wall off (sequester) both the injurious agent and the injuredtissue. Inflammation is notably associated with influx of leukocytesand/or neutrophil chemotaxis. Inflammation can result from infectionwith pathogenic organisms and viruses and from noninfectious means suchas trauma or reperfusion following myocardial infarction or stroke,immune response to foreign antigen, and autoimmune responses.Accordingly, inflammatory disorders amenable to treatment with Formula Iand II compounds encompass disorders associated with reactions of thespecific defense system as well as with reactions of the nonspecificdefense system.

“Specific defense system” refers to the component of the immune systemthat reacts to the presence of specific antigens. Examples ofinflammation resulting from a response of the specific defense systeminclude the classical response to foreign antigens, autoimmune diseases,and delayed type hypersensitivity response mediated by T-cells. Chronicinflammatory diseases, the rejection of solid transplanted tissue andorgans, e.g., kidney and bone marrow transplants, and graft versus hostdisease (GVHD), are further examples of inflammatory reactions of thespecific defense system.

The term “nonspecific defense system” as used herein refers toinflammatory disorders that are mediated by leukocytes that areincapable of immunological memory (e.g., granulocytes, and macrophages).Examples of inflammation that result, at least in part, from a reactionof the nonspecific defense system include inflammation associated withconditions such as adult (acute) respiratory distress syndrome (ARDS) ormultiple organ injury syndromes; reperfusion injury; acuteglomerulonephritis; reactive arthritis; dermatoses with acuteinflammatory components; acute purulent meningitis or other centralnervous system inflammatory disorders such as stroke; thermal injury;inflammatory bowel disease; granulocyte transfusion associatedsyndromes; and cytokine-induced toxicity.

“Autoimmune disease” as used herein refers to any group of disorders inwhich tissue injury is associated with humoral or cell-mediatedresponses to the body's own constituents.

“Allergic disease” as used herein refers to any symptoms, tissue damage,or loss of tissue function resulting from allergy. “Arthritic disease”as used herein refers to any disease that is characterized byinflammatory lesions of the joints attributable to a variety ofetiologies. “Dermatitis” as used herein refers to any of a large familyof diseases of the skin that are characterized by inflammation of theskin attributable to a variety of etiologies. “Transplant rejection” asused herein refers to any immune reaction directed against graftedtissue, such as organs or cells (e.g., bone marrow), characterized by aloss of function of the grafted and surrounding tissues, pain, swelling,leukocytosis, and thrombocytopenia. The therapeutic methods of thepresent invention include methods for the treatment of disordersassociated with inflammatory cell activation.

“Inflammatory cell activation” refers to the induction by a stimulus(including, but not limited to, cytokines, antigens or auto-antibodies)of a proliferative cellular response, the production of solublemediators (including but not limited to cytokines, oxygen radicals,enzymes, prostanoids, or vasoactive amines), or cell surface expressionof new or increased numbers of mediators (including, but not limited to,major histocompatability antigens or cell adhesion molecules) ininflammatory cells (including but not limited to monocytes, macrophages,T lymphocytes, B lymphocytes, granulocytes (i.e., polymorphonuclearleukocytes such as neutrophils, basophils, and eosinophils), mast cells,dendritic cells, Langerhans cells, and endothelial cells). It will beappreciated by persons skilled in the art that the activation of one ora combination of these phenotypes in these cells can contribute to theinitiation, perpetuation, or exacerbation of an inflammatory disorder.

The term “NSAID” is an acronym for “non-steroidal anti-inflammatorydrug” and is a therapeutic agent with analgesic, antipyretic (loweringan elevated body temperature and relieving pain without impairingconsciousness) and, in higher doses, with anti-inflammatory effects(reducing inflammation). The term “non-steroidal” is used to distinguishthese drugs from steroids, which (among a broad range of other effects)have a similar eicosanoid-depressing, anti-inflammatory action. Asanalgesics, NSAIDs are unusual in that they are non-narcotic. NSAIDsinclude aspirin, ibuprofen, and naproxen. NSAIDs are usually indicatedfor the treatment of acute or chronic conditions where pain andinflammation are present. NSAIDs are generally indicated for thesymptomatic relief of the following conditions: rheumatoid arthritis,osteoarthritis, inflammatory arthropathies (e.g. ankylosing spondylitis,psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhoea,metastatic bone pain, headache and migraine, postoperative pain,mild-to-moderate pain due to inflammation and tissue injury, pyrexia,ileus, and renal colic. Most NSAIDs act as non-selective inhibitors ofthe enzyme cyclooxygenase, inhibiting both the cyclooxygenase-1 (COX-1)and cyclooxygenase-2 (COX-2) isoenzymes. Cyclooxygenase catalyzes theformation of prostaglandins and thromboxane from arachidonic acid(itself derived from the cellular phospholipid bilayer by phospholipaseA₂). Prostaglandins act (among other things) as messenger molecules inthe process of inflammation. COX-2 inhibitors include celecoxib,etoricoxib, lumiracoxib, parecoxib, rofecoxib, rofecoxib, andvaldecoxib.

The terms “cancer” refers to or describe the physiological condition inmammals that is typically characterized by unregulated cell growth. A“tumor” comprises one or more cancerous cells. Examples of cancerinclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer (e.g., epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer (“NSCLC”), adenocarcinoma of the lung and squamous carcinomaof the lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head andneck cancer.

“Hematological malignancies” (British spelling “Haematological”malignancies) are the types of cancer that affect blood, bone marrow,and lymph nodes. As the three are intimately connected through theimmune system, a disease affecting one of the three will often affectthe others as well: although lymphoma is a disease of the lymph nodes,it often spreads to the bone marrow, affecting the blood. Hematologicalmalignancies are malignant neoplasms (“cancer”), and they are generallytreated by specialists in hematology and/or oncology. In some centers“Hematology/oncology” is a single subspecialty of internal medicinewhile in others they are considered separate divisions (there are alsosurgical and radiation oncologists). Not all hematological disorders aremalignant (“cancerous”); these other blood conditions may also bemanaged by a hematologist. Hematological malignancies may derive fromeither of the two major blood cell lineages: myeloid and lymphoid celllines. The myeloid cell line normally produces granulocytes,erythrocytes, thrombocytes, macrophages and mast cells; the lymphoidcell line produces B, T, NK and plasma cells. Lymphomas, lymphocyticleukemias, and myeloma are from the lymphoid line, while acute andchronic myelogenous leukemia, myelodysplastic syndromes andmyeloproliferative diseases are myeloid in origin. Leukemias includeAcute lymphoblastic leukemia (ALL), Acute myelogenous leukemia (AML),Chronic lymphocytic leukemia (CLL), Chronic myelogenous leukemia (CML),Acute monocytic leukemia (AMOL) and small lymphocytic lymphoma (SLL).Lymphomas include Hodgkin's lymphomas (all four subtypes) andNon-Hodgkin's lymphomas (all subtypes).

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to: alkylatingagents, antimetabolites, spindle poison plant alkaloids,cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,photosensitizers, and kinase inhibitors. Chemotherapeutic agents includecompounds used in “targeted therapy” and conventional chemotherapy.Examples of chemotherapeutic agents include: erlotinib (TARCEVA®,Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU(fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®,Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin(cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin(CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide,CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2,HPPD, and rapamycin.

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin(folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib(TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs),gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11,Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478,AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib(GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa andcyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, calicheamicin gamma1I, calicheamicin omegaI1 (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin,marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (NAVELBINE0); novantrone; teniposide;edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche);ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid; andpharmaceutically acceptable salts, acids and derivatives of any of theabove.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipidkinase inhibitors; (vi) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, suchas oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitorssuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” aretherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®,Genentech), tositumomab (Bexxar, Corixia), and the antibody drugconjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential aschemotherapeutic agents in combination with the Btk inhibitors of theinvention include: alemtuzumab, apolizumab, aselizumab, atlizumab,bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumabmertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab,daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab,fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab,labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab,motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab,ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab,pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab,urtoxazumab, and visilizumab.

A “metabolite” is a product produced through metabolism in the body of aspecified compound or salt thereof. Metabolites of a compound may beidentified using routine techniques known in the art and theiractivities determined using tests such as those described herein. Suchproducts may result for example from the oxidation, reduction,hydrolysis, amidation, deamidation, esterification, deesterification,enzymatic cleavage, and the like, of the administered compound.Accordingly, the invention includes metabolites of compounds of theinvention, including compounds produced by a process comprisingcontacting a Formula I or II compound of this invention with a mammalfor a period of time sufficient to yield a metabolic product thereof.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand 1 or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or 1 meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity. Enantiomers may be separated from a racemic mixture bya chiral separation method, such as supercritical fluid chromatography(SFC). Assignment of configuration at chiral centers in separatedenantiomers may be tentative, and depicted in Table 1 structures forillustrative purposes, while stereochemical determination awaits, suchas x-ray crystallographic data.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

The term “pharmaceutically acceptable salts” denotes salts which are notbiologically or otherwise undesirable. Pharmaceutically acceptable saltsinclude both acid and base addition salts. The phrase “pharmaceuticallyacceptable” indicates that the substance or composition must becompatible chemically and/or toxicologically, with the other ingredientscomprising a formulation, and/or the mammal being treated therewith.

The term “pharmaceutically acceptable acid addition salt” denotes thosepharmaceutically acceptable salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,carbonic acid, phosphoric acid, and organic acids selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic, and sulfonic classes of organic acids such as formic acid,acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid,pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid,ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamicacid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonicacid “mesylate”, ethanesulfonic acid, p-toluenesulfonic acid, andsalicyclic acid.

The term “pharmaceutically acceptable base addition salt” denotes thosepharmaceutically acceptable salts formed with an organic or inorganicbase. Examples of acceptable inorganic bases include sodium, potassium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, andaluminum salts. Salts derived from pharmaceutically acceptable organicnontoxic bases includes salts of primary, secondary, and tertiaryamines, substituted amines including naturally occurring substitutedamines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, and polyamine resins

A “solvate” refers to an association or complex of one or more solventmolecules and a compound of the invention. Examples of solvents thatform solvates include, but are not limited to, water, isopropanol,ethanol, methanol, DMSO, ethylacetate, acetic acid, and ethanolamine.

The term “EC₅₀” is the half maximal effective concentration” and denotesthe plasma concentration of a particular compound required for obtaining50% of the maximum of a particular effect in vivo.

The term “Ki” is the inhibition constant and denotes the absolutebinding affinity of a particular inhibitor to a receptor. It is measuredusing competition binding assays and is equal to the concentration wherethe particular inhibitor would occupy 50% of the receptors if nocompeting ligand (e.g. a radioligand) was present. Ki values can beconverted logarithmically to pKi values (−log Ki), in which highervalues indicate exponentially greater potency.

The term “IC₅₀” is the half maximal inhibitory concentration and denotesthe concentration of a particular compound required for obtaining 50%inhibition of a biological process in vitro. IC₅₀ values can beconverted logarithmically to pIC₅₀ values (−log IC₅₀), in which highervalues indicate exponentially greater potency. The IC₅₀ value is not anabsolute value but depends on experimental conditions e.g.concentrations employed, and can be converted to an absolute inhibitionconstant (Ki) using the Cheng-Prusoff equation (Biochem. Pharmacol.(1973) 22:3099). Other percent inhibition parameters, such as IC₇₀,IC₉₀, etc., may be calculated.

The terms “compound of this invention,” and “compounds of the presentinvention” and “compounds of Formula I” include compounds of Formulas Iand stereoisomers, geometric isomers, tautomers, solvates, metabolites,and pharmaceutically acceptable salts and prodrugs thereof.

Any formula or structure given herein, including Formula I and IIcompounds, is also intended to represent hydrates, solvates, andpolymorphs of such compounds, and mixtures thereof.

Any formula or structure given herein, including Formula I and IIcompounds, is also intended to represent unlabeled forms as well asisotopically labeled forms of the compounds. Isotopically labeledcompounds have structures depicted by the formulas given herein exceptthat one or more atoms are replaced by an atom having a selected atomicmass or mass number. Examples of isotopes that can be incorporated intocompounds of the invention include isotopes of hydrogen, carbon,nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as, but notlimited to 2H (deuterium, D), 3H (tritium), 11C, 13C, 14C, 15N, 18F,31P, 32P, 35S, 36Cl, and 125I. Various isotopically labeled compounds ofthe present invention, for example those into which radioactive isotopessuch as 3H, 13C, and 14C are incorporated. Such isotopically labelledcompounds may be useful in metabolic studies, reaction kinetic studies,detection or imaging techniques, such as positron emission tomography(PET) or single-photon emission computed tomography (SPECT) includingdrug or substrate tissue distribution assays, or in radioactivetreatment of patients. Deuterium labelled or substituted therapeuticcompounds of the invention may have improved DMPK (drug metabolism andpharmacokinetics) properties, relating to distribution, metabolism, andexcretion (ADME). Substitution with heavier isotopes such as deuteriummay afford certain therapeutic advantages resulting from greatermetabolic stability, for example increased in vivo half-life or reduceddosage requirements. An 18F labeled compound may be useful for PET orSPECT studies. Isotopically labeled compounds of this invention andprodrugs thereof can generally be prepared by carrying out theprocedures disclosed in the schemes or in the examples and preparationsdescribed below by substituting a readily available isotopically labeledreagent for a non-isotopically labeled reagent. Further, substitutionwith heavier isotopes, particularly deuterium (i.e., 2H or D) may affordcertain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements or an improvement in therapeutic index. It is understoodthat deuterium in this context is regarded as a substituent in thecompound of the formula (I). The concentration of such a heavierisotope, specifically deuterium, may be defined by an isotopicenrichment factor. In the compounds of this invention any atom notspecifically designated as a particular isotope is meant to representany stable isotope of that atom. Unless otherwise stated, when aposition is designated specifically as “H” or “hydrogen”, the positionis understood to have hydrogen at its natural abundance isotopiccomposition. Accordingly, in the compounds of this invention any atomspecifically designated as a deuterium (D) is meant to representdeuterium.

8-Fluorophthalazin-1(2H)-One Compounds

The present invention provides 8-fluorophthalazin-1(2h)-one compounds ofFormula I, including Formulas Ia-If, and pharmaceutical formulationsthereof, which are potentially useful in the treatment of diseases,conditions and/or disorders modulated by Btk kinase:

or stereoisomers, tautomers, or pharmaceutically acceptable saltsthereof, wherein:

X¹ is CR¹ or N;

X² is CR² or N;

X³ is CR³ or N;

where one or two of X¹, X², and X³ are N;

R¹, R² and R³ are independently selected from H, F, Cl, CN, —CH₃,—CH₂CH₃, —CH₂OH, —CH₂F, —CHF₂, —CF₃, —CH₂CH₂OH, —NH₂, —NHCH₃, —N(CH₃)₂,—OH, —OCH₃, —OCH₂CH₃, and —OCH₂CH₂OH;

R⁴ is selected from H, F, Cl, CN, —CH₂OH, —CH(CH₃)OH, —C(CH₃)₂OH,—CH(CF₃)OH, —CH₂F, —CHF₂, —CH₂CHF₂, —CF₃, —C(O)NH₂, —C(O)NHCH₃,—C(O)N(CH₃)₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHC(O)CH₃, —OH, —OCH₃, —OCH₂CH₃,—OCH₂CH₂OH, cyclopropyl, cyclopropylmethyl, 1-hydroxycyclopropyl,imidazolyl, pyrazolyl, 3-hydroxy-oxetan-3-yl, oxetan-3-yl, andazetidin-1-yl;

R⁵ is selected from —CH₃, —CH₂CH₃, —CH₂OH, —CH₂F, —CHF₂, —CF₃, —CN, and—CH₂CH₂OH;

or two R⁵ groups form a 3-, 4-, 5-, or 6-membered carbocyclic orheterocyclic ring;

or an R⁵ group and an R⁷ group form a 3-, 4-, 5-, or 6-memberedcarbocyclic or heterocyclic ring;

n is 0, 1, 2, 3, or 4;

R⁶ is selected from H, —CH₃, —CH₂CH₃, —CH₂CH₂OH, —CH₂F, —CHF₂, —CF₃,—NH₂, —NHCH₃, —N(CH₃)₂, —OH, —OCH₃, —OCH₂CH₃, and —OCH₂CH₂OH;

R⁷ is selected from H, —CH₃, —S(O)₂CH₃, cyclopropyl, azetidin-3-yl,oxetan-3-yl, and morpholin-4-yl;

Z¹ is CR⁸ or N, where R⁸ is selected from H, F, Cl, —CH₃, —CH₂CH₃,—CH₂CH₂OH, —NH₂, —NHCH₃, —N(CH₃)₂, —OH, —OCH₃, —OCH₂CH₃, and —OCH₂CH₂OH;

Z² is CR⁹ or N, where R⁹ is selected from H, —CH₃, —CH₂CH₃, and—CH₂CH₂OH; and

Y¹ and Y² are independently selected from CH and N, where Y¹ and Y² arenot each N.

Exemplary embodiments of Formula I compounds include compounds ofFormulas Ia-If:

Exemplary embodiments of Formula I compounds include wherein X¹ is N, X²is CR², and X³ is CR³.

Exemplary embodiments of Formula I compounds include wherein X¹ is CR¹,X² is N, and X³ is CR³.

Exemplary embodiments of Formula I compounds include wherein X¹ is CR¹,X² is CR², and X³ is N.

Exemplary embodiments of Formula I compounds include wherein: X¹ and X³are N, X¹ and X² are N, or X² and X³ are N.

Exemplary embodiments of Formula I compounds include wherein X² is CR²,and R² is F.

Exemplary embodiments of Formula I compounds include wherein X¹ and X³are CH.

Exemplary embodiments of Formula I compounds include wherein R⁴ is—CH₂OH.

Exemplary embodiments of Formula I compounds include wherein R⁵ is —CH₃,and n is 1 or 2.

Exemplary embodiments of Formula I compounds include wherein R⁷ isoxetan-3-yl.

Exemplary embodiments of Formula I compounds include wherein Y¹ is CH.

Exemplary embodiments of Formula I compounds include wherein Y² is CH.

Exemplary embodiments of Formula I compounds include wherein Y¹ is N.

Exemplary embodiments of Formula I compounds include wherein Y² is N.

Exemplary embodiments of Formula I compounds include wherein Z¹ is CH.

Exemplary embodiments of Formula I compounds include wherein Z² is CH.

Exemplary embodiments of Formula I compounds include wherein Z¹ is N.

Exemplary embodiments of Formula I compounds include wherein Z² is N.

The present invention also provides 8-fluorophthalazin-1(2h)-onecompounds of Formula II, which are potentially useful in the treatmentof diseases, conditions and/or disorders modulated by Btk kinase:

or stereoisomers, tautomers, or pharmaceutically acceptable saltsthereof, wherein:

X¹ is CR¹ or N;

X² is CR² or N;

X³ is CR³ or N;

where one or two of X¹, X², and X³ are N;

Y¹ and Y² are independently selected from CH and N, where Y¹ and Y² arenot each N;

R¹, R² and R³ are independently selected from H, F, Cl, CN, —CH₃,—CH₂CH₃, —CH₂OH, —CH₂F, —CHF₂, —CF₃, —CH₂CH₂OH, —NH₂, —NHCH₃, —N(CH₃)₂,—OH, —OCH₃, —OCH₂CH₃, and —OCH₂CH₂OH;

R⁴ is selected from H, F, Cl, CN, —CH₂OH, —CH(CH₃)OH, —C(CH₃)₂OH,—CH(CF₃)OH, —CH₂F, —CHF₂, —CH₂CHF₂, —CF₃, —C(O)NH₂, —C(O)NHCH₃,—C(O)N(CH₃)₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHC(O)CH₃, —OH, —OCH₃, —OCH₂CH₃,—OCH₂CH₂OH, —OP(O)(OH)₂, cyclopropyl, cyclopropylmethyl,1-hydroxycyclopropyl, imidazolyl, pyrazolyl, 3-hydroxy-oxetan-3-yl,oxetan-3-yl, and azetidin-1-yl;

R⁶ is selected from H, —CH₃, —CH₂CH₃, —CH₂CH₂OH, —CH₂F, —CHF₂, —CF₃,—NH₂, —NHCH₃, —N(CH₃)₂, —OH, —OCH₃, —OCH₂CH₃, and —OCH₂CH₂OH;

R⁸ is selected from C₆-C₂₀ aryl, C₃-C₁₂ carbocyclyl, C₂-C₂₀heterocyclyl, C₁-C₂₀ heteroaryl, —(C₆-C₂₀ aryl)-(C₂-C₂₀ heterocyclyl),—(C₁-C₂₀ heteroaryl)-(C₂-C₂₀ heterocyclyl), —(C₁-C₂₀ heteroaryl)-(C₂-C₂₀heterocyclyl)-(C₂-C₂₀ heterocyclyl), —(C₁-C₂₀ heteroaryl)-(C₂-C₂₀heterocyclyl)-(C₁-C₆ alkyl), —(C₁-C₂₀ heteroaryl)-(C₁-C₆ alkyl), and—(C₁-C₂₀ heteroaryl)-C(═O)—(C₂-C₂₀ heterocyclyl); where aryl,carbocyclyl, heterocyclyl, and heteroaryl are optionally substitutedwith one or more groups selected from F, Cl, Br, I, CN, —CH₃, —CH₂CH₃,CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH₂OH, —CH₂OCH₃, —C(CH₃)₂OH, —CH(OH)CH(CH₃)₂,—C(CH₃)₂CH₂OH, —CH₂CH₂SO₂CH₃, —CH₂OP(O)(OH)₂, —C(CH₃)₂CONH₂, —CH₂OCH₃,—CH₂CH₂OH, —CH₂CH₂OCH₃, —CH₂F, —CHF₂, —CF₃, —CH₂CF₃, —CH₂CHF₂,—CH(CH₃)CN, —C(CH₃)₂CN, —CH₂CN, CO₂H, —CO₂CH₃, —CO₂C(CH₃)₃,—COCH(OH)CH₃, —C(O)CH₃, —C(O)CH₂CH₃, —C(O)CH(CH₃)₂, —C(O)NH₂,—C(O)NHCH₃, —C(O)N(CH₃)₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHC(O)CH₃,—N(CH₃)COCH₃, —NHS(O)₂CH₃, —N(CH₃)C(CH₃)₂CONH₂, —N(CH₃)CH₂CH₂S(O)₂CH₃,—NO₂, ═O, —OCH₂CH₂N(CH₃)₂, —OP(O)(OH)₂, —OH, —OCH₃, —OCH₂CH₃,—OCH₂CH₂OCH₃, —OCH₂CH₂OH, —S(O)₂N(CH₃)₂, —SCH₃, —S(O)₂CH₃, —S(O)₃H,cyclopropyl, oxetanyl, azetidinyl, 1-methylazetidin-3-yl)oxy,N-methyl-N-oxetan-3-ylamino, azetidin-1-ylmethyl, and morpholino.

Exemplary embodiments of Formula II compounds include wherein R⁸ is—(C₁-C₂₀ heteroaryl)-(C₂-C₂₀ heterocyclyl).

Exemplary embodiments of Formula II compounds include wherein R⁸ ispyridinyl.

Exemplary embodiments of Formula II compounds include wherein R⁸ is-(pyridinyl)-(piperazinyl).

Exemplary embodiments of Formula II compounds include wherein R⁸ isC₁-C₂₀ heteroaryl.

Exemplary embodiments of Formula II compounds include wherein R⁸ isselected from:

pyrimidinyl,

6,7-dihydro-4H-thiazolo[5,4-c]pyridin-2-yl,

5-(morpholine-4-carbonyl)-2-pyridyl,

pyrazolyl,

thiazolyl,

6,7-dihydro-4H-pyrazolo[1,5-a]pyrazin-2-yl,

oxazolyl,

isoxazolyl,

imidazolyl,

5-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-yl,

1,2,3-triazolyl,

4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine,

pyrazinyl, and

5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-2-yl.

Exemplary embodiments of Formula II compounds include wherein R⁸ is

where R⁹ is selected from H, —CH₃, —CH₂OCH₃, —CH₂CH₃, —CH(CH₃)₂,—CH₂CH₂OH, —CH₂CH₂OCH₃, —CH₂F, —CHF₂, —CF₃, —CH₂CF₃, —CH₂CHF₂,—CH(CH₃)CN, —C(CH₃)₂CN, —CH₂CN, —CH₂CH₂CN, —C(O)CH₃, —C(O)CH₂CH₃,—C(O)CH(CH₃)₂, —NH₂, —NHCH₃, —N(CH₃)₂, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂OH,cyclopropyl, cyclopropylmethyl, oxetanyl and oxetanylmethyl.

Exemplary embodiments of Formula II compounds include wherein X¹ is N,X² is CR², and X³ is CR³.

Exemplary embodiments of Formula II compounds include wherein X¹ is CR¹,X² is N, and X³ is CR³.

Exemplary embodiments of Formula II compounds include wherein X¹ is CR¹,X² is CR², and X³ is N.

Exemplary embodiments of Formula II compounds include wherein X¹ and X³are N, X¹ and X² are N, or X² and X³ are N.

Exemplary embodiments of Formula II compounds include wherein X² is CR²,and R² is F.

Exemplary embodiments of Formula II compounds include wherein X¹ and X³are CH.

Exemplary embodiments of Formula II compounds include wherein R⁴ is—CH₂OH.

The Formula I and II compounds of the invention may contain asymmetricor chiral centers, and therefore exist in different stereoisomericforms. It is intended that all stereoisomeric forms of the compounds ofthe invention, including but not limited to, diastereomers, enantiomersand atropisomers, as well as mixtures thereof such as racemic mixtures,form part of the present invention.

In addition, the present invention embraces all diastereomers, includingcis-trans (geometric) and conformational isomers. For example, if aFormula I compound incorporates a double bond or a fused ring, the cis-and trans-forms, as well as mixtures thereof, are embraced within thescope of the invention.

In the structures shown herein, where the stereochemistry of anyparticular chiral atom is not specified, then all stereoisomers arecontemplated and included as the compounds of the invention. Wherestereochemistry is specified by a solid wedge or dashed linerepresenting a particular configuration, then that stereoisomer is sospecified and defined.

The compounds of the present invention may exist in unsolvated as wellas solvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like, and it is intended that the inventionembrace both solvated and unsolvated forms.

The compounds of the present invention may also exist in differenttautomeric forms, and all such forms are embraced within the scope ofthe invention. The term “tautomer” or “tautomeric form” refers tostructural isomers of different energies which are interconvertible viaa low energy barrier. For example, proton tautomers (also known asprototropic tautomers) include interconversions via migration of aproton, such as keto-enol and imine-enamine isomerizations. Valencetautomers include interconversions by reorganization of some of thebonding electrons.

Biological Evaluation

The relative efficacies of Formula I compounds as inhibitors of anenzyme activity (or other biological activity) can be established bydetermining the concentrations at which each compound inhibits theactivity to a predefined extent and then comparing the results.Typically, the preferred determination is the concentration thatinhibits 50% of the activity in a biochemical assay, i.e., the 50%inhibitory concentration or “IC₅₀”. Determination of IC₅₀ values can beaccomplished using conventional techniques known in the art. In general,an IC₅₀ can be determined by measuring the activity of a given enzyme inthe presence of a range of concentrations of the inhibitor under study.The experimentally obtained values of enzyme activity then are plottedagainst the inhibitor concentrations used. The concentration of theinhibitor that shows 50% enzyme activity (as compared to the activity inthe absence of any inhibitor) is taken as the IC₅₀ value. Analogously,other inhibitory concentrations can be defined through appropriatedeterminations of activity. For example, in some settings it can bedesirable to establish a 90% inhibitory concentration, i.e., IC₉₀, etc.

Formula I compounds were tested by a standard biochemical Btk KinaseAssay (Example 901).

A general procedure for a standard cellular Btk Kinase Assay that can beused to test Formula I compounds is a Ramos Cell Btk Assay (Example902).

A standard cellular B-cell proliferation assay can be used to testFormula I compounds with B-cells purified from spleen of Balb/c mice(Example 903).

A standard T cell proliferation assay can be used to test Formula Icompounds with T-cells purified from spleen of Balb/c mice (Example904).

A CD86 Inhibition assay can be conducted on Formula I compounds for theinhibition of B cell activity using total mouse splenocytes purifiedfrom spleens of 8-16 week old Balb/c mice (Example 905).

A B-ALL Cell Survival Assay can be conducted on Formula I compounds tomeasure the number of viable B-ALL cells in culture (Example 906).

A CD69 Whole Blood Assay can be conducted on Formula I compounds todetermine the ability of compounds to inhibit the production of CD69 byB lymphocytes in human whole blood activated by crosslinking surface IgMwith goat F(ab′)2 anti-human IgM (Example 907). CD69 is a type II C-typelectin involved in lymphocyte migration and cytokine secretion. CD69expression represents one of the earliest available indicators ofleukocyte activation and its rapid induction occurs throughtranscriptional activation (Vazquez et al (2009) Jour. of ImmunologyPublished Oct. 19, 2009, doi:10.4049/jimmunol.0900839).Concentration-dependent inhibition of antigen receptor stimulation byselective Btk inhibitors induces cell surface expression of thelymphocyte activation marker CD69 (Honigberg et al (2010) Proc. Natl.Acad. Sci. 107(29):13075-13080). Thus, CD69 inhibition by selective Btkinhibitors may be correlated with therapeutic efficacy of certain B-celldisorders. The CD69 Hu Blood FACS IC70 values are displayed forexemplary Formula I compounds in Tables 1 and 2.

The cytotoxic or cytostatic activity of Formula I exemplary compoundscan be measured by: establishing a proliferating mammalian tumor cellline in a cell culture medium, adding a Formula I compound, culturingthe cells for a period from about 6 hours to about 5 days; and measuringcell viability (Example 908). Cell-based in vitro assays are used tomeasure viability, i.e. proliferation (IC₅₀), cytotoxicity (EC₅₀), andinduction of apoptosis (caspase activation) and may be useful inpredicting clinical efficacy against hematological malignancies andsolid tumors.

The in vitro potency of the combinations of Formula I compounds withchemotherapeutic agents can be measured by the cell proliferation assayof Example 908; the CellTiter-Glo® Luminescent Cell Viability Assay,commercially available from Promega Corp., Madison, Wis. Thishomogeneous assay method is based on the recombinant expression ofColeoptera luciferase (U.S. Pat. No. 5,583,024; U.S. Pat. No. 5,674,713;U.S. Pat. No. 5,700,670) and determines the number of viable cells inculture based on quantitation of the ATP present, an indicator ofmetabolically active cells (Crouch et al (1993) J. Immunol. Meth.160:81-88; U.S. Pat. No. 6,602,677). The CellTiter-Glo® Assay wasconducted in 96 or 384 well format, making it amenable to automatedhigh-throughput screening (HTS) (Cree et al (1995) AntiCancer Drugs6:398-404). The homogeneous assay procedure involves adding the singlereagent (CellTiter-Glo® Reagent) directly to cells cultured inserum-supplemented medium. Cell washing, removal of medium and multiplepipetting steps are not required. The system detects as few as 15cells/well in a 384-well format in 10 minutes after adding reagent andmixing.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The amount of ATP is directly proportional to the number ofcells present in culture. The CellTiter-Glo® Assay generates a“glow-type” luminescent signal, produced by the luciferase reaction,which has a half-life generally greater than five hours, depending oncell type and medium used. Viable cells are reflected in relativeluminescence units (RLU). The substrate, Beetle Luciferin, isoxidatively decarboxylated by recombinant firefly luciferase withconcomitant conversion of ATP to AMP and generation of photons. Theextended half-life eliminates the need to use reagent injectors andprovides flexibility for continuous or batch mode processing of multipleplates. This cell proliferation assay can be used with various multiwellformats, e.g. 96 or 384 well format. Data can be recorded by luminometeror CCD camera imaging device. The luminescence output is presented asrelative light units (RLU), measured over time.

The anti-proliferative efficacy of Formula I exemplary compounds andcombinations with chemotherapeutic agents are measured by theCellTiter-Glo® Assay (Example 908) against certain hematological tumorcell lines. EC₅₀ values are established for the tested compounds andcombinations.

Exemplary Formula I compounds in Tables 1 and 2 were made,characterized, and tested for inhibition of Btk according to the methodsof this invention, and have the following structures and correspondingnames (ChemDraw Ultra, Version 9.0.1, and ChemBioDraw, Version 11.0,CambridgeSoft Corp., Cambridge Mass.). Where more than one name isassociated with a Formula I compound or intermediate, the chemicalstructure shall define the compound.

TABLE 1 CD69 Hu Blood Mol FACS No. Structure IUPAC_Name Weight IC70 101

6-tert-butyl-8-fluoro-2-(2- (hydroxymethyl)-3-(1- methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2- yl-amino)-6-oxo-1,6- dihydropyridin-3-yl)phenyl)phthalazin- 1(2H)-one 623.30 0.024 102

6-tert-butyl-8-fluoro-2-(5- fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(4- (oxetan-3-yl)piperazin-1- yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3- yl)phenyl)phthalazin- 1(2H)-one 684 0.012 103

6-tert-butyl-8-fluoro-2-(3- (hydroxymethyl)-4-(1-methyl-5-(5-(4-(oxetan-3- yl)piperazin-1-yl)pyridin- 2-ylamino)-6-oxo-1,6dihydropyridin-3- yl)pyridin-2-yl)phthalazin- 1(2H)-one 667 0.013 104

(S)-6-tert-butyl-8-fluoro-2- (5-fluoro-2- (hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4- (oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6- oxo-1,6-dihydropyridin-3- yl)phenyl)phthalazin-1(2H)-one 697.32 0.0329 105

(S)-6-tert-butyl-8-fluoro-2- (3-(hydroxymethyl)-4-(1-methyl-5-(5-(2-methyl-4- (oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6- oxo-1,6-dihydropyridin-3-yl)pyridin-2-yl)phthalazin- 1(2H)one 680.32 0.0060 106

(R)-6-tert-butyl-8-fluoro-2- (3-(hydroxymethyl)-4-(1-methyl-5-(5-(2-methyl-4- (oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6- oxo-1,6-dihydropyridin-3-yl)pyridin-2-yl)phthalazin- 1(2H)-one 680.32 107

(R)-6-tert-butyl-8-fluoro-2- (5-fluoro-2- (hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4- (oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6- oxo-1,6-dihydropyridin-3- yl)phenyl)phthalazin-1(2H)-one 697.32 0.0263 108

6-tert-butyl-2-(3-(5-(5- ((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1- yl)pyridin-2-ylamino)-1- methyl-6-oxo-1,6-dihydropyridin-3-yl)-5- fluoro-2- (hydroxymethyl)phenyl)-8-fluorophthalazin-1(2H)- one 711.33 109

(S)-6-tert-butyl-8-fluoro-2- (4-(hydroxymethyl)-5-(1-methyl-5-(5-(2-methyl-4- (oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6- oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)phthalazin- 1(2H)-one 680.32 0.012 110

6-tert-butyl-2-(4-(5-(5- ((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1- yl)pyridin-2-ylamino)-1- methyl-6-oxo-1,6-dihydropyridin-3-yl)-3- (hydroxymethyl)pyridin-2-yl)-8-fluorophthalazin- 1(2H)-one 694.34 0.0059 111

(S)-6-tert-butyl-2-(3-(5-(5- (2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin- 2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3- yl)-5-fluoro-2- (hydroxymethyl)phenyl)-8-fluorophthalazin-1(2H)- one 711.33 0.0374 112

(S)-6-tert-butyl-2-(4-(5-(5- (2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin- 2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3- yl)-3- (hydroxymethyl)pyridin-2-yl)-8-fluorophthalazin- 1(2H)-one 694.34 0.0329

TABLE 2 CD69 Hu Blood FACS No. Structure IUPAC_Name (IC70) 113

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-6-oxo-5-(pyrimidin-4- ylamino)-3-pyridyl]-2- pyridyl]phthalazin-1-one0.0823 114

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[(2-methylpyrimidin-4- yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one 0.114 115

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[(5-methyl-6,7-dihydro-4H- thiazolo[5,4-c]pyridin-2-yl)amino]-6-oxo-3-pyridyl]-2- pyridyl]phthalazin-1-one 0.0732 116

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[[5-(morpholine-4- carbonyl)-2-pyridyl]amino]-6-oxo-pyridazin-3-yl]-2- pyridyl]phthalazin-1-one 0.0935 117

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[[5-[(2S)-2-methyl-4- (oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-6-oxo- pyridazin-3-yl]-2- pyridyl]phthalazin-1-one0.0165 118

6-tert-butyl-2-[4-[5-[(1-ethyl- 5-methyl-pyrazol-3-yl)amino]-1-methyl-6-oxo-3-pyridyl]-3- (hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one 0.112 119

6-tert-butyl-2-[4-[5-[(1,5- dimethylpyrazol-3-yl)amino]-1-methyl-6-oxo-3-pyridyl]-3- (hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one 0.0247 120

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[(5-methylthiazol-2- yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one 0.139 121

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[(5-methyl-1H-pyrazol-3- yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one 0.0793 122

6-tert-butyl-2-[4-[5-[(5-ethyl- 1-methyl-pyrazol-3-yl)amino]-1-methyl-6-oxo-3-pyridyl]-3- (hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one 0.0375 123

6-tert-butyl-2-[4-[6-[(1- ethylpyrazol-4-yl)amino]-4-methyl-5-oxo-pyrazin-2-yl]-3- (hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one 0.156 124

2-[4-[5-[(5-acetyl-6,7-dihydro- 4H-pyrazolo[1,5-a]pyrazin-2-yl)amino]-1-methyl-6-oxo-3- pyridyl]-3-(hydroxymethyl)-2-pyridyl]-6-tert-butyl-8-fluoro- phthalazin-1-one 0.0415 125

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[(5-methyloxazol-2- yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one 0.521 126

6-tert-butyl-2-[4-[5-[(5- cyclopropyl-1-methyl-pyrazol-3-yl)amino]-1-methyl-6-oxo- 3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8-fluoro- phthalazin-1-one 0.0445 127

6-tert-butyl-2-[4-[5-[(1,5- dimethylpyrazol-3-yl)amino]-1-methyl-6-oxo-pyridazin-3- yl]-3-(hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin- 1-one 0.115 128

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[(5-methyl-6,7-dihydro-4H- pyrazolo[1,5-a]pyrazin-2-yl)amino]-6-oxo-3-pyridyl]-2- pyridyl]phthalazin-1-one 0.0272 129

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[(5-methylisoxazol-3- yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one 0.0488 130

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[(1-methylimidazol-4- yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one 0.152 131

6-tert-butyl-2-[4-[5-(6,7- dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1- methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8- fluoro-phthalazin-1-one 0.0171 132

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl- 5-[[5-[(3R)-3-methylmorpholine-4- carbonyl]-2-pyridyl]amino]-6- oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one 0.014 133

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[5-[[5-(2-methoxyethyl)-6,7-dihydro- 4H-pyrazolo[1,5-a]pyrazin-2-yl]amino]-1-methyl-6-oxo-3- pyridyl]-2-pyridyl]phthalazin- 1-one 0.0145134

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-6-oxo-5-[[5-(2,2,2- trifluoroethyl)-6,7-dihydro-4H-pyrazolo[1,5-a]pyrazin-2- yl]amino]-3-pyridyl]-2-pyridyl]phthalazin-1-one 0.0216 135

6-tert-butyl-2-[4-[5-[[5-(2,2- difluoroethyl)-6,7-dihydro-4H-pyrazolo[1,5-a]pyrazin-2- yl]amino]-1-methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2- pyridyl]-8-fluoro-phthalazin- 1-one 0.02136

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl- 5-[[5-[(3S)-3-methylmorpholine-4- carbonyl]-2-pyridyl]amino]-6- oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one 0.0145 137 138

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[5-[[5-[(2S)-2-methyl-4-(oxetan-3- yl)piperazin-1-yl]-2-pyridyl]amino]-6-oxo-1H- pyridin-3-yl]-2- pyridyl]phthalazin-1-one0.0124 139

6-tert-butyl-2-[4-[5-[(6,6- dimethyl-4,7- dihydropyrazolo[5,1-c][1,4]oxazin-2-yl)amino]-1- methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8- fluoro-phthalazin-1-one 0.0616 140

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[(1-methylpyrazol-3- yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one 0.068 141

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[(5-methylisoxazol-3- yl)amino]-6-oxo-pyridazin-3-yl]-2-pyridyl]phthalazin-1-one 0.295 142

6-tert-butyl-2-[4-[5-[(1- ethylpyrazol-3-yl)amino]-1-methyl-6-oxo-3-pyridyl]-3- (hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one 0.168 143

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[5-[[5-(methoxymethyl)-1-methyl- pyrazol-3-yl]amino]-1-methyl-6-oxo-3-pyridyl]-2- pyridyl]phthalazin-1-one 0.0131 144

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[[1-methyl-5-(pyrrolidine-1- carbonyl)pyrazol-3-yl]amino]-6-oxo-3-pyridyl]-2- pyridyl]phthalazin-1-one 0.0203 145

6-tert-butyl-2-[4-[5-[[1-(2,2- difluoroethyl)-5-methyl-pyrazol-3-yl]amino]-1-methyl- 6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8- fluoro-phthalazin-1-one 0.163 146

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[(5-methyl-6-oxo-4,7- dihydropyrazolo[1,5-a]pyrazin-2-yl)amino]-6-oxo- 3-pyridyl]-2- pyridyl]phthalazin-1-one0.0209 147

6-tert-butyl-8-fluoro-2-[4-[5- [[5-(3-hydroxyazetidin-1-yl)-2-pyridyl]amino]-1-methyl-6- oxo-3-pyridyl]-3- (hydroxymethyl)-2-pyridyl]phthalazin-1-one 0.0333 148

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[[5-(2-methylpropanoyl)- 6,7-dihydro-4H-pyrazolo[1,5-a]pyrazin-2-yl]amino]-6-oxo- 3-pyridyl]-2- pyridyl]phthalazin-1-one0.0139 149

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[(1-methyltriazol-4- yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one 0.093 150

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[(4-methyl-6,7-dihydro-4H- pyrazolo[5,1-c][1,4]oxazin-2-yl)amino]-6-oxo-3-pyridyl]-2- pyridyl]phthalazin-1-one 0.0428 151

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[5-[(5-isopropyl-6-oxo-4,7- dihydropyrazolo[1,5- a]pyrazin-2-yl)amino]-1-methyl-6-oxo-3-pyridyl]-2- pyridyl]phthalazin-1-one 0.0141 152

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-6-oxo-5-(1H-pyrazol-3- ylamino)-3-pyridyl]-2- pyridyl]phthalazin-1-one0.0459 153

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[(5-methyl-6,7-dihydro-4H- pyrazolo[1,5-a]pyrazin-2-yl)amino]-6-oxo-pyridazin-3- yl]-2-pyridyl]phthalazin-1-one 0.0596 154

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[(3-methyltriazol-4- yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one 155

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[[5-(oxetan-3-yl)-6,7- dihydro-4H-pyrazolo[1,5-a]pyrazin-2-yl]amino]-6-oxo- pyridazin-3-yl]-2- pyridyl]phthalazin-1-one0.0266 156

2-[6-[[5-[2-(6-tert-butyl-8- fluoro-1-oxo-phthalazin-2-yl)-3-(hydroxymethyl)-4-pyridyl]- 1-methyl-2-oxo-3-pyridyl]amino]-3-pyridyl]-2- methyl-propanenitrile 0.031 157

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-5-[(5-methylisothiazol-3- yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one 0.157 158

6-tert-butyl-2-[4-[5-[(5- ethylisoxazol-3-yl)amino]-1-methyl-6-oxo-3-pyridyl]-3- (hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one 0.0667 159

6-tert-butyl-2-[4-[5-[[2- (difluoromethyl)triazol-4-yl]amino]-1-methyl-6-oxo-3- pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin- 1-one 0.208 160

6-tert-butyl-8-fluoro-2-[3- (hydroxymethyl)-4-[1-methyl-6-oxo-5-(pyrazin-2-ylamino)- 3-pyridyl]-2- pyridyl]phthalazin-1-one0.116 161

[2-(6-tert-butyl-8-fluoro-1- oxo-phthalazin-2-yl)-4-[1-methyl-5-[(5-methylisoxazol- 3-yl)amino]-6-oxo-3-pyridyl]-3-pyridyl]methyl dihydrogen phosphate 162

6-tert-butyl-2-[4-[5-(5,6- dihydro-4H-pyrrolo[1,2-b]pyrazol-2-ylamino)-1- methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8- fluoro-phthalazin-1-one

Administration of Formula I Compounds

The compounds of the invention may be administered by any routeappropriate to the condition to be treated. Suitable routes includeoral, parenteral (including subcutaneous, intramuscular, intravenous,intraarterial, intradermal, intrathecal and epidural), transdermal,rectal, nasal, topical (including buccal and sublingual), vaginal,intraperitoneal, intrapulmonary and intranasal. For localimmunosuppressive treatment, the compounds may be administered byintralesional administration, including perfusing or otherwisecontacting the graft with the inhibitor before transplantation. It willbe appreciated that the preferred route may vary with for example thecondition of the recipient. Where the compound is administered orally,it may be formulated as a pill, capsule, tablet, etc. with apharmaceutically acceptable carrier or excipient. Where the compound isadministered parenterally, it may be formulated with a pharmaceuticallyacceptable parenteral vehicle and in a unit dosage injectable form, asdetailed below.

A dose to treat human patients may range from about 10 mg to about 1000mg of a Formula I or II compound. A typical dose may be about 100 mg toabout 300 mg of the compound. A dose may be administered once a day(QID), twice per day (BID), or more frequently, depending on thepharmacokinetic and pharmacodynamic properties, including absorption,distribution, metabolism, and excretion of the particular compound. Inaddition, toxicity factors may influence the dosage and administrationregimen. When administered orally, the pill, capsule, or tablet may beingested daily or less frequently for a specified period of time. Theregimen may be repeated for a number of cycles of therapy.

Methods of Treatment with Formula I and II Compounds

Formula I and II compounds of the present invention are useful fortreating a human or animal patient suffering from a disease or disorderarising from abnormal cell growth, function or behavior associated withBtk kinase such as an immune disorder, cardiovascular disease, viralinfection, inflammation, a metabolism/endocrine disorder or aneurological disorder, may thus be treated by a method comprising theadministration thereto of a compound of the present invention as definedabove. A human or animal patient suffering from cancer may also betreated by a method comprising the administration thereto of a compoundof the present invention as defined above. The condition of the patientmay thereby be improved or ameliorated.

Formula I and II compounds may be useful for in vitro, in situ, and invivo diagnosis or treatment of mammalian cells, organisms, or associatedpathological conditions, such as systemic and local inflammation,immune-inflammatory diseases such as rheumatoid arthritis, immunesuppression, organ transplant rejection, allergies, ulcerative colitis,Crohn's disease, dermatitis, asthma, systemic lupus erythematosus,Sjögren's Syndrome, multiple sclerosis, scleroderma/systemic sclerosis,idiopathic thrombocytopenic purpura (ITP), anti-neutrophil cytoplasmicantibodies (ANCA) vasculitis, chronic obstructive pulmonary disease(COPD), psoriasis, and for general joint protective effects.

Methods of the invention also include treating such diseases asarthritic diseases, such as rheumatoid arthritis, monoarticulararthritis, osteoarthritis, gouty arthritis, spondylitis; Behcet disease;sepsis, septic shock, endotoxic shock, gram negative sepsis, grampositive sepsis, and toxic shock syndrome; multiple organ injurysyndrome secondary to septicemia, trauma, or hemorrhage; ophthalmicdisorders such as allergic conjunctivitis, vernal conjunctivitis,uveitis, and thyroid-associated ophthalmopathy; eosinophilic granuloma;pulmonary or respiratory disorders such as asthma, chronic bronchitis,allergic rhinitis, ARDS, chronic pulmonary inflammatory disease (e.g.,chronic obstructive pulmonary disease), silicosis, pulmonarysarcoidosis, pleurisy, alveolitis, vasculitis, emphysema, pneumonia,bronchiectasis, and pulmonary oxygen toxicity; reperfusion injury of themyocardium, brain, or extremities; fibrosis such as cystic fibrosis;keloid formation or scar tissue formation; atherosclerosis; autoimmunediseases, such as systemic lupus erythematosus (SLE), autoimmunethyroiditis, multiple sclerosis, some forms of diabetes, and Reynaud'ssyndrome; and transplant rejection disorders such as GVHD and allograftrejection; chronic glomerulonephritis; inflammatory bowel diseases suchas chronic inflammatory bowel disease (CIBD), Crohn's disease,ulcerative colitis, and necrotizing enterocolitis; inflammatorydermatoses such as contact dermatitis, atopic dermatitis, psoriasis, orurticaria; fever and myalgias due to infection; central or peripheralnervous system inflammatory disorders such as meningitis, encephalitis,and brain or spinal cord injury due to minor trauma; Sjögren's syndrome;diseases involving leukocyte diapedesis; alcoholic hepatitis; bacterialpneumonia; antigen-antibody complex mediated diseases; hypovolemicshock; Type I diabetes mellitus; acute and delayed hypersensitivity;disease states due to leukocyte dyscrasia and metastasis; thermalinjury; granulocyte transfusion-associated syndromes; andcytokine-induced toxicity.

Methods of the invention also include treating cancer selected frombreast, ovary, cervix, prostate, testis, genitourinary tract, esophagus,larynx, glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma,lung, epidermoid carcinoma, large cell carcinoma, non-small cell lungcarcinoma (NSCLC), small cell carcinoma, lung adenocarcinoma, bone,colon, adenoma, pancreas, adenocarcinoma, thyroid, follicular carcinoma,undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma,sarcoma, bladder carcinoma, liver carcinoma and biliary passages, kidneycarcinoma, pancreatic, myeloid disorders, lymphoma, hairy cells, buccalcavity, naso-pharyngeal, pharynx, lip, tongue, mouth, small intestine,colon-rectum, large intestine, rectum, brain and central nervous system,Hodgkin's, leukemia, bronchus, thyroid, liver and intrahepatic bileduct, hepatocellular, gastric, glioma/glioblastoma, endometrial,melanoma, kidney and renal pelvis, urinary bladder, uterine corpus,uterine cervix, multiple myeloma, acute myelogenous leukemia, chronicmyelogenous leukemia, lymphocytic leukemia, chronic lymphoid leukemia(CLL), myeloid leukemia, oral cavity and pharynx, non-Hodgkin lymphoma,melanoma, and villous colon adenoma.

The methods of the invention can have utility in treating subjects whoare or can be subject to reperfusion injury, i.e., injury resulting fromsituations in which a tissue or organ experiences a period of ischemiafollowed by reperfusion. The term “ischemia” refers to localized tissueanemia due to obstruction of the inflow of arterial blood. Transientischemia followed by reperfusion characteristically results inneutrophil activation and transmigration through the endothelium of theblood vessels in the affected area. Accumulation of activatedneutrophils in turn results in generation of reactive oxygenmetabolites, which damage components of the involved tissue or organ.This phenomenon of “reperfusion injury” is commonly associated withconditions such as vascular stroke (including global and focalischemia), hemorrhagic shock, myocardial ischemia or infarction, organtransplantation, and cerebral vasospasm. To illustrate, reperfusioninjury occurs at the termination of cardiac bypass procedures or duringcardiac arrest when the heart, once prevented from receiving blood,begins to reperfuse. It is expected that inhibition of Btk activity mayresult in reduced amounts of reperfusion injury in such situations.

Pharmaceutical Formulations

In order to use a compound of this invention for the therapeutictreatment of mammals including humans, it is normally formulated inaccordance with standard pharmaceutical practice as a pharmaceuticalcomposition. According to this aspect of the invention there is provideda pharmaceutical composition comprising a compound of this invention inassociation with a pharmaceutically acceptable diluent or carrier.

A typical formulation is prepared by mixing a compound of the presentinvention and a carrier, diluent or excipient. Suitable carriers,diluents and excipients are well known to those skilled in the art andinclude materials such as carbohydrates, waxes, water soluble and/orswellable polymers, hydrophilic or hydrophobic materials, gelatin, oils,solvents, water and the like. The particular carrier, diluent orexcipient used will depend upon the means and purpose for which thecompound of the present invention is being applied. Solvents aregenerally selected based on solvents recognized by persons skilled inthe art as safe (GRAS) to be administered to a mammal. In general, safesolvents are non-toxic aqueous solvents such as water and othernon-toxic solvents that are soluble or miscible in water. Suitableaqueous solvents include water, ethanol, propylene glycol, polyethyleneglycols (e.g., PEG 400, PEG 300), etc. and mixtures thereof. Theformulations may also include one or more buffers, stabilizing agents,surfactants, wetting agents, lubricating agents, emulsifiers, suspendingagents, preservatives, antioxidants, opaquing agents, glidants,processing aids, colorants, sweeteners, perfuming agents, flavoringagents and other known additives to provide an elegant presentation ofthe drug (i.e., a compound of the present invention or pharmaceuticalcomposition thereof) or aid in the manufacturing of the pharmaceuticalproduct (i.e., medicament).

The formulations may be prepared using conventional dissolution andmixing procedures. For example, the bulk drug substance (i.e., compoundof the present invention or stabilized form of the compound (e.g.,complex with a cyclodextrin derivative or other known complexationagent) is dissolved in a suitable solvent in the presence of one or moreof the excipients described above. The compound of the present inventionis typically formulated into pharmaceutical dosage forms to provide aneasily controllable dosage of the drug and to enable patient compliancewith the prescribed regimen.

The pharmaceutical composition (or formulation) for application may bepackaged in a variety of ways depending upon the method used foradministering the drug. Generally, an article for distribution includesa container having deposited therein the pharmaceutical formulation inan appropriate form. Suitable containers are well known to those skilledin the art and include materials such as bottles (plastic and glass),sachets, ampoules, plastic bags, metal cylinders, and the like. Thecontainer may also include a tamper-proof assemblage to preventindiscreet access to the contents of the package. In addition, thecontainer has deposited thereon a label that describes the contents ofthe container. The label may also include appropriate warnings.

Pharmaceutical formulations of the compounds of the present inventionmay be prepared for various routes and types of administration. Forexample, a compound of Formula I or II having the desired degree ofpurity may optionally be mixed with pharmaceutically acceptablediluents, carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.), in the formof a lyophilized formulation, milled powder, or an aqueous solution.Formulation may be conducted by mixing at ambient temperature at theappropriate pH, and at the desired degree of purity, withphysiologically acceptable carriers, i.e., carriers that are non-toxicto recipients at the dosages and concentrations employed. The pH of theformulation depends mainly on the particular use and the concentrationof compound, but may range from about 3 to about 8. Formulation in anacetate buffer at pH 5 is a suitable embodiment.

The compound ordinarily can be stored as a solid composition, alyophilized formulation or as an aqueous solution.

The pharmaceutical compositions of the invention will be formulated,dosed and administered in a fashion, i.e., amounts, concentrations,schedules, course, vehicles and route of administration, consistent withgood medical practice. Factors for consideration in this context includethe particular disorder being treated, the particular mammal beingtreated, the clinical condition of the individual patient, the cause ofthe disorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of the compound to be administered will be governed by suchconsiderations, and is the minimum amount necessary to ameliorate, ortreat the hyperproliferative disorder.

As a general proposition, the initial pharmaceutically effective amountof the inhibitor administered parenterally per dose will be in the rangeof about 0.01-100 mg/kg, namely about 0.1 to 20 mg/kg of patient bodyweight per day, with the typical initial range of compound used being0.3 to 15 mg/kg/day.

Acceptable diluents, carriers, excipients and stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Theactive pharmaceutical ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations of compounds of Formula I or II may beprepared. Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing acompound of Formula I or II, which matrices are in the form of shapedarticles, e.g., films, or microcapsules. Examples of sustained-releasematrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate) and poly-D-(−)-3-hydroxybutyric acid.

The formulations include those suitable for the administration routesdetailed herein. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. Techniques and formulations generally are found inRemington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).Such methods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

Formulations of a compound of Formula I or II suitable for oraladministration may be prepared as discrete units such as pills,capsules, cachets or tablets each containing a predetermined amount of acompound of Formula I or II. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface active ordispersing agent. Molded tablets may be made by molding in a suitablemachine a mixture of the powdered active ingredient moistened with aninert liquid diluent. The tablets may optionally be coated or scored andoptionally are formulated so as to provide slow or controlled release ofthe active ingredient therefrom. Tablets, troches, lozenges, aqueous oroil suspensions, dispersible powders or granules, emulsions, hard orsoft capsules, e.g., gelatin capsules, syrups or elixirs may be preparedfor oral use. Formulations of compounds of Formula I or II intended fororal use may be prepared according to any method known to the art forthe manufacture of pharmaceutical compositions and such compositions maycontain one or more agents including sweetening agents, flavoringagents, coloring agents and preserving agents, in order to provide apalatable preparation. Tablets containing the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipient which aresuitable for manufacture of tablets are acceptable. These excipients maybe, for example, inert diluents, such as calcium or sodium carbonate,lactose, calcium or sodium phosphate; granulating and disintegratingagents, such as maize starch, or alginic acid; binding agents, such asstarch, gelatin or acacia; and lubricating agents, such as magnesiumstearate, stearic acid or talc. Tablets may be uncoated or may be coatedby known techniques including microencapsulation to delay disintegrationand adsorption in the gastrointestinal tract and thereby provide asustained action over a longer period. For example, a time delaymaterial such as glyceryl monostearate or glyceryl distearate alone orwith a wax may be employed.

For treatment of the eye or other external tissues, e.g., mouth andskin, the formulations are preferably applied as a topical ointment orcream containing the active ingredient(s) in an amount of, for example,0.075 to 20% w/w. When formulated in an ointment, the active ingredientsmay be employed with either a paraffinic or a water-miscible ointmentbase. Alternatively, the active ingredients may be formulated in a creamwith an oil-in-water cream base. If desired, the aqueous phase of thecream base may include a polyhydric alcohol, i.e., an alcohol having twoor more hydroxyl groups such as propylene glycol, butane 1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400)and mixtures thereof. The topical formulations may desirably include acompound which enhances absorption or penetration of the activeingredient through the skin or other affected areas. Examples of suchdermal penetration enhancers include dimethyl sulfoxide and relatedanalogs. The oily phase of the emulsions of this invention may beconstituted from known ingredients in a known manner. While the phasemay comprise merely an emulsifier, it desirably comprises a mixture ofat least one emulsifier with a fat or an oil or with both a fat and anoil. Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabilizer. It is also preferredto include both an oil and a fat. Together, the emulsifier(s) with orwithout stabilizer(s) make up the so-called emulsifying wax, and the waxtogether with the oil and fat make up the so-called emulsifying ointmentbase which forms the oily dispersed phase of the cream formulations.Emulsifiers and emulsion stabilizers suitable for use in the formulationof the invention include Tween® 60, Span® 80, cetostearyl alcohol,benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodiumlauryl sulfate.

Aqueous suspensions of Formulas I and II compounds contain the activematerials in admixture with excipients suitable for the manufacture ofaqueous suspensions. Such excipients include a suspending agent, such assodium carboxymethylcellulose, croscarmellose, povidone,methylcellulose, hydroxypropyl methylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing orwetting agents such as a naturally occurring phosphatide (e.g.,lecithin), a condensation product of an alkylene oxide with a fatty acid(e.g., polyoxyethylene stearate), a condensation product of ethyleneoxide with a long chain aliphatic alcohol (e.g.,heptadecaethyleneoxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol anhydride(e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension mayalso contain one or more preservatives such as ethyl or n-propylp-hydroxybenzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose or saccharin.

The pharmaceutical compositions of compounds of Formulas I and II may bein the form of a sterile injectable preparation, such as a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, such as a solution in 1,3-butanediol or prepared as alyophilized powder. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile fixed oils may conventionally be employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid may likewise be used in the preparationof injectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain approximately 1 to 1000 mg of active material compounded with anappropriate and convenient amount of carrier material which may varyfrom about 5 to about 95% of the total compositions (weight:weight). Thepharmaceutical composition can be prepared to provide easily measurableamounts for administration. For example, an aqueous solution intendedfor intravenous infusion may contain from about 3 to 500 μg of theactive ingredient per milliliter of solution in order that infusion of asuitable volume at a rate of about 30 mL/hr can occur.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is preferably present in suchformulations in a concentration of about 0.5 to 20% w/w, for exampleabout 0.5 to 10% w/w, for example about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of 0.1 to 500 microns (includingparticle sizes in a range between 0.1 and 500 microns in incrementsmicrons such as 0.5, 1, 30 microns, 35 microns, etc.), which isadministered by rapid inhalation through the nasal passage or byinhalation through the mouth so as to reach the alveolar sacs. Suitableformulations include aqueous or oily solutions of the active ingredient.Formulations suitable for aerosol or dry powder administration may beprepared according to conventional methods and may be delivered withother therapeutic agents such as compounds heretofore used in thetreatment or prophylaxis disorders as described below.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

The formulations may be packaged in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water, for injection immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefore. Veterinary carriers are materials useful for thepurpose of administering the composition and may be solid, liquid orgaseous materials which are otherwise inert or acceptable in theveterinary art and are compatible with the active ingredient. Theseveterinary compositions may be administered parenterally, orally or byany other desired route.

Combination Therapy

The compounds of Formulas I and II may be employed alone or incombination with other therapeutic agents for the treatment of a diseaseor disorder described herein, such as inflammation or ahyperproliferative disorder (e.g., cancer). In certain embodiments, acompound of Formula I or II is combined in a pharmaceutical combinationformulation, or dosing regimen as combination therapy, with anadditional, second therapeutic compound that has anti-inflammatory oranti-hyperproliferative properties or that is useful for treating aninflammation, immune-response disorder, or hyperproliferative disorder(e.g., cancer). The additional therapeutic may be an anti-inflammatoryagent, an immunomodulatory agent, chemotherapeutic agent, anapoptosis-enhancer, a neurotropic factor, an agent for treatingcardiovascular disease, an agent for treating liver disease, ananti-viral agent, an agent for treating blood disorders, an agent fortreating diabetes, and an agent for treating immunodeficiency disorders.The second therapeutic agent may be an NSAID anti-inflammatory agent.The second therapeutic agent may be a chemotherapeutic agent. The secondcompound of the pharmaceutical combination formulation or dosing regimenpreferably has complementary activities to the compound of Formula I orII such that they do not adversely affect each other. Such compounds aresuitably present in combination in amounts that are effective for thepurpose intended. In one embodiment, a composition of this inventioncomprises a compound of Formula I or II, or a stereoisomer, tautomer,solvate, metabolite, or pharmaceutically acceptable salt or prodrugthereof, in combination with a therapeutic agent such as an NSAID.

The combination therapy may be administered as a simultaneous orsequential regimen. When administered sequentially, the combination maybe administered in two or more administrations. The combinedadministration includes coadministration, using separate formulations ora single pharmaceutical formulation, and consecutive administration ineither order, wherein preferably there is a time period while both (orall) active agents simultaneously exert their biological activities.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the newly identified agent and other therapeutic agents ortreatments.

The combination therapy may provide “synergy” and prove “synergistic”,i.e., the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect may be attained when the compounds are administered or deliveredsequentially, e.g., by different injections in separate syringes,separate pills or capsules, or separate infusions. In general, duringalternation therapy, an effective dosage of each active ingredient isadministered sequentially, i.e., serially, whereas in combinationtherapy, effective dosages of two or more active ingredients areadministered together.

In a particular embodiment of therapy, a compound of Formula I or II, ora stereoisomer, tautomer, solvate, metabolite, or pharmaceuticallyacceptable salt or prodrug thereof, may be combined with othertherapeutic, hormonal or antibody agents such as those described herein,as well as combined with surgical therapy and radiotherapy. Combinationtherapies according to the present invention thus comprise theadministration of at least one compound of Formula I or II, or astereoisomer, tautomer, solvate, metabolite, or pharmaceuticallyacceptable salt or prodrug thereof, and the use of at least one othercancer treatment method. The amounts of the compound(s) of Formula I orII, and the other pharmaceutically active therapeutic agent(s) and therelative timings of administration will be selected in order to achievethe desired combined therapeutic effect.

Metabolites of Compounds of Formulas I and II

Also falling within the scope of this invention are the in vivometabolic products of Formulas I and II compounds described herein. Suchproducts may result for example from the oxidation, reduction,hydrolysis, amidation, deamidation, esterification, deesterification,enzymatic cleavage, and the like, of the administered compound.Accordingly, the invention includes metabolites of compounds of FormulaI and II, including compounds produced by a process comprisingcontacting a compound of this invention with a mammal for a period oftime sufficient to yield a metabolic product thereof.

Metabolite products typically are identified by preparing aradiolabelled (e.g., ¹⁴C or ³H) isotope of a compound of the invention,administering it parenterally in a detectable dose (e.g., greater thanabout 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, orto man, allowing sufficient time for metabolism to occur (typicallyabout 30 seconds to 30 hours) and isolating its conversion products fromthe urine, blood or other biological samples. These products are easilyisolated since they are labeled (others are isolated by the use ofantibodies capable of binding epitopes surviving in the metabolite). Themetabolite structures are determined in conventional fashion, e.g., byMS, LC/MS or NMR analysis. In general, analysis of metabolites is donein the same way as conventional drug metabolism studies well known tothose skilled in the art. The metabolite products, so long as they arenot otherwise found in vivo, are useful in diagnostic assays fortherapeutic dosing of the compounds of the invention.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture, or“kit”, containing materials useful for the treatment of the diseases anddisorders described above is provided. In one embodiment, the kitcomprises a container comprising a compound of Formula I or II, or astereoisomer, tautomer, solvate, metabolite, or pharmaceuticallyacceptable salt or prodrug thereof. The kit may further comprise a labelor package insert on or associated with the container. The term “packageinsert” is used to refer to instructions customarily included incommercial packages of therapeutic products, that contain informationabout the indications, usage, dosage, administration, contraindicationsand/or warnings concerning the use of such therapeutic products.Suitable containers include, for example, bottles, vials, syringes,blister pack, etc. The container may be formed from a variety ofmaterials such as glass or plastic. The container may hold a compound ofFormula I or II, or a formulation thereof which is effective fortreating the condition and may have a sterile access port (for example,the container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). At least oneactive agent in the composition is a compound of Formula I or II. Thelabel or package insert indicates that the composition is used fortreating the condition of choice, such as cancer. In addition, the labelor package insert may indicate that the patient to be treated is onehaving a disorder such as a hyperproliferative disorder,neurodegeneration, cardiac hypertrophy, pain, migraine or aneurotraumatic disease or event. In one embodiment, the label or packageinserts indicates that the composition comprising a compound of FormulaI or II can be used to treat a disorder resulting from abnormal cellgrowth. The label or package insert may also indicate that thecomposition can be used to treat other disorders. Alternatively, oradditionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

The kit may further comprise directions for the administration of thecompound of Formula I or II and, if present, the second pharmaceuticalformulation. For example, if the kit comprises a first compositioncomprising a compound of Formula I or II, and a second pharmaceuticalformulation, the kit may further comprise directions for thesimultaneous, sequential or separate administration of the first andsecond pharmaceutical compositions to a patient in need thereof.

In another embodiment, the kits are suitable for the delivery of solidoral forms of a compound of Formula I or II, such as tablets orcapsules. Such a kit preferably includes a number of unit dosages. Suchkits can include a card having the dosages oriented in the order oftheir intended use. An example of such a kit is a “blister pack”.Blister packs are well known in the packaging industry and are widelyused for packaging pharmaceutical unit dosage forms. If desired, amemory aid can be provided, for example in the form of numbers, letters,or other markings or with a calendar insert, designating the days in thetreatment schedule in which the dosages can be administered.

According to one embodiment, a kit may comprise (a) a first containerwith a compound of Formula I or II contained therein; and optionally (b)a second container with a second pharmaceutical formulation containedtherein, wherein the second pharmaceutical formulation comprises asecond compound with anti-hyperproliferative activity. Alternatively, oradditionally, the kit may further comprise a third container comprisinga pharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

In certain other embodiments wherein the kit comprises a composition ofFormula I or II and a second therapeutic agent, the kit may comprise acontainer for containing the separate compositions such as a dividedbottle or a divided foil packet, however, the separate compositions mayalso be contained within a single, undivided container. Typically, thekit comprises directions for the administration of the separatecomponents. The kit form is particularly advantageous when the separatecomponents are preferably administered in different dosage forms (e.g.,oral and parenteral), are administered at different dosage intervals, orwhen titration of the individual components of the combination isdesired by the prescribing physician.

Preparation of Formulas I and II Compounds

Compounds of Formula I and II may be synthesized by synthetic routesthat include processes analogous to those well-known in the chemicalarts, particularly in light of the description contained herein, andthose for other heterocycles described in: Comprehensive HeterocyclicChemistry II, Editors Katritzky and Rees, Elsevier, 1997, e.g. Volume 3;Liebigs Annalen der Chemie, (9):1910-16, (1985); Helvetica Chimica Acta,41:1052-60, (1958); Arzneimittel-Forschung, 40(12):1328-31, (1990), eachof which are expressly incorporated by reference. Starting materials aregenerally available from commercial sources such as Aldrich Chemicals(Milwaukee, Wis.) or are readily prepared using methods well known tothose skilled in the art (e.g., prepared by methods generally describedin Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v.1-23, Wiley, N.Y. (1967-2006 ed.), or Beilsteins Handbuch derorganischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin, includingsupplements (also available via the Beilstein online database).

Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing Formula I and IIcompounds and necessary reagents and intermediates are known in the artand include, for example, those described in R. Larock, ComprehensiveOrganic Transformations, VCH Publishers (1989); T. W. Greene and P. G.M. Wuts, Protective Groups in Organic Synthesis, 3^(rd) Ed., John Wileyand Sons (1999); and L. Paquette, ed., Encyclopedia of Reagents forOrganic Synthesis, John Wiley and Sons (1995) and subsequent editionsthereof.

Compounds of Formula I and II may be prepared singly or as compoundlibraries comprising at least 2, for example 5 to 1,000 compounds, or 10to 100 compounds. Libraries of compounds of Formula I and II may beprepared by a combinatorial ‘split and mix’ approach or by multipleparallel syntheses using either solution phase or solid phase chemistry,by procedures known to those skilled in the art. Thus according to afurther aspect of the invention there is provided a compound librarycomprising at least 2 compounds, or pharmaceutically acceptable saltsthereof.

The Figures and Examples provide exemplary methods for preparing FormulaI and II compounds. Those skilled in the art will appreciate that othersynthetic routes may be used to synthesize the Formula I and IIcompounds. Although specific starting materials and reagents aredepicted and discussed in the Figures and Examples, other startingmaterials and reagents can be easily substituted to provide a variety ofderivatives and/or reaction conditions. In addition, many of theexemplary compounds prepared by the described methods can be furthermodified in light of this disclosure using conventional chemistry wellknown to those skilled in the art.

In preparing compounds of Formulas I, protection of remote functionality(e.g., primary or secondary amine) of intermediates may be necessary.The need for such protection will vary depending on the nature of theremote functionality and the conditions of the preparation methods.Suitable amino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz) and9-fluorenylmethyleneoxycarbonyl (Fmoc). The need for such protection isreadily determined by one skilled in the art. For a general descriptionof protecting groups and their use, see T. W. Greene, Protective Groupsin Organic Synthesis, John Wiley & Sons, New York, 1991.

Experimental procedures, intermediates and reagents useful for usefulfor the preparation of Formula I and II compounds may be found in US2012/0010191, “PYRIDONE AND AZA-PYRIDONE COMPOUNDS AND METHODS OF USE”,filed 6 May 2011, which is incorporated by reference in its entirety.

FIGS. 1-8 describe the synthesis of exemplary embodiments of Formula Ior II, more fully described in the Examples 101-112, and may be usefulfor the preparation of other Formula I and II compounds.

General Preparative Procedures

The Suzuki-type coupling reaction is useful to form carbon-carbon bondsto attach the rings of Formula I and II compounds and intermediates A-3(Suzuki (1991) Pure Appl. Chem. 63:419-422; Miyaura and Suzuki (1979)Chem. Reviews 95(7):2457-2483; Suzuki (1999) J. Organometal. Chem.576:147-168). Suzuki coupling is a palladium mediated cross couplingreaction of a heteroarylhalide, such as B-2 or B-4, with a boronic acidsuch as A-1 or A-2. For example, B-2 may be combined with about 1.5equivalents of4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane), anddissolved in about 3 equivalents of sodium carbonate as a 1 molarsolution in water and an equal volume of acetonitrile. A catalyticamount, or more, of a low valent palladium reagent, such asbis(triphenylphosphine)palladium(II) dichloride, is added. In some casespotassium acetate is used in place of sodium carbonate to adjust the pHof the aqueous layer. The reaction is then heated to about 140-150° C.under pressure in a microwave reactor (Biotage AB, Uppsala, Sweden) for10 to 30 minutes. The contents are extracted with ethyl acetate, oranother organic solvent. After evaporation of the organic layer theboron ester A-1 may be purified on silica or by reverse phase HPLC.Substituents are as defined, or protected forms or precursors thereof.Likewise, bromide intermediate B-4 can be boronylated to give A-2.

Suzuki coupling of B-2 and A-2, or of A-1 and B-4, gives Formula Icompound or intermediate A-3. Boronic ester (or acid) (1.5 eq) A-1 orA-2, and a palladium catalyst such asbis(triphenylphosphine)palladium(II) chloride (0.05 eq) is added to amixture of halo intermediate (1 eq) B-2 or B-4 in acetonitrile and 1 Mof sodium carbonate aqueous solution (equal volume as acetonitrile). Thereaction mixture is heated to about 150° C. in a microwave for about 15min. LC/MS indicates when the reaction is complete. Water is added tothe mixture, and the precipitated product is filtered and purified byHPLC to yield the product A-3. Substituents are as defined, or protectedforms or precursors thereof.

A variety of palladium catalysts can be used during the Suzuki couplingstep. Various low valent, Pd(II) and Pd(0) catalysts may be used in theSuzuki coupling reaction, including PdCl2(PPh₃)₂, Pd(t-Bu)₃, PdCl₂ dppfCH₂Cl₂, Pd(PPh₃)₄, Pd(OAc)/PPh₃, Cl₂Pd[(Pet₃)]₂, Pd(DIPHOS)₂,Cl₂Pd(Bipy), [PdCl(Ph₂PCH₂PPh₂)]₂, Cl₂Pd[P(o-tol)₃]₂,Pd₂(dba)₃/P(o-tol)₃, Pd₂(dba)/P(furyl)₃, Cl₂Pd[P(furyl)₃]₂,Cl₂Pd(PMePh₂)₂, Cl₂Pd[P(4-F-Ph)₃]₂, Cl₂Pd[P(C₆F₆)₃]₂,Cl₂Pd[P(2-COOH-Ph)(Ph)₂]₂, Cl₂Pd[P(4-COOH-Ph)(Ph)₂]₂, and encapsulatedcatalysts Pd EnCat™ 30, Pd EnCat™ TPP30, and Pd(II)EnCat™ BINAP30 (US2004/0254066).

The Buchwald reaction is useful to aminate 6-bromo intermediates B-1(Wolf and Buchwald (2004) Org. Synth Coll. Vol. 10:423; Paul et al(1994) Jour. Amer. Chem. Soc. 116:5969-5970). To a solution of halointermediate B-1 in DMF is added the appropriate piperazinyl-pyridinylor piperazinyl-pyrimidinyl amine (200 mol %), Cs₂CO₃ (50 mol %),Pd₂(dba)₃ (5 mol %), and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene(Xantphos, CAS Reg. No. 161265-03-8, 10 mol %). The reaction is heatedto about 110° C. under pressure in a microwave reactor (Biotage AB,Uppsala, Sweden) for about 30 min. The resulting solution isconcentrated in vacuo to give B-2. Other palladium catalysts andphosphine ligands may be useful.

N-Heteroaryl amide intermediates B-4 can also be prepared under Buchwaldconditions with cyclic amide intermediates (R⁷) such as6-tert-butyl-8-fluorophthalazin-1(2H)-one 101h and heteroaryl dibromidesB-3.

Methods of Separation

In the methods of preparing Formula I and II compounds, it may beadvantageous to separate reaction products from one another and/or fromstarting materials. The desired products of each step or series of stepsis separated and/or purified to the desired degree of homogeneity by thetechniques common in the art. Typically such separations involvemultiphase extraction, crystallization from a solvent or solventmixture, distillation, sublimation, or chromatography. Chromatographycan involve any number of methods including, for example: reverse-phaseand normal phase; size exclusion; ion exchange; high, medium and lowpressure liquid chromatography methods and apparatus; small scaleanalytical; simulated moving bed (SMB) and preparative thin or thicklayer chromatography, as well as techniques of small scale thin layerand flash chromatography.

Another class of separation methods involves treatment of a mixture witha reagent selected to bind to or render otherwise separable a desiredproduct, unreacted starting material, reaction by product, or the like.Such reagents include adsorbents or absorbents such as activated carbon,molecular sieves, ion exchange media, or the like. Alternatively, thereagents can be acids in the case of a basic material, bases in the caseof an acidic material, binding reagents such as antibodies, bindingproteins, selective chelators such as crown ethers, liquid/liquid ionextraction reagents (LIX), or the like. Selection of appropriate methodsof separation depends on the nature of the materials involved, such as,boiling point and molecular weight in distillation and sublimation,presence or absence of polar functional groups in chromatography,stability of materials in acidic and basic media in multiphaseextraction, and the like.

Diastereomeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods well known to those skilled in the art, such as bychromatography and/or fractional crystallization. Enantiomers can beseparated by converting the enantiomeric mixture into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,chiral auxiliary such as a chiral alcohol or Mosher's acid chloride),separating the diastereomers and converting (e.g., hydrolyzing) theindividual diastereoisomers to the corresponding pure enantiomers. Also,some of the compounds of the present invention may be atropisomers(e.g., substituted biaryls) and are considered as part of thisinvention. Enantiomers can also be separated by use of a chiral HPLCcolumn.

A single stereoisomer, e.g., an enantiomer, substantially free of itsstereoisomer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (Eliel, E. and Wilen, S. “Stereochemistry of OrganicCompounds,” John Wiley & Sons, Inc., New York, 1994; Lochmuller, C. H.,(1975) J. Chromatogr., 113(3):283-302). Racemic mixtures of chiralcompounds of the invention can be separated and isolated by any suitablemethod, including: (1) formation of ionic, diastereomeric salts withchiral compounds and separation by fractional crystallization or othermethods, (2) formation of diastereomeric compounds with chiralderivatizing reagents, separation of the diastereomers, and conversionto the pure stereoisomers, and (3) separation of the substantially pureor enriched stereoisomers directly under chiral conditions. See: “DrugStereochemistry, Analytical Methods and Pharmacology,” Irving W. Wainer,Ed., Marcel Dekker, Inc., New York (1993).

Under method (1), diastereomeric salts can be formed by reaction ofenantiomerically pure chiral bases such as brucine, quinine, ephedrine,strychnine, α-methyl-β-phenylethylamine (amphetamine), and the like withasymmetric compounds bearing acidic functionality, such as carboxylicacid and sulfonic acid. The diastereomeric salts may be induced toseparate by fractional crystallization or ionic chromatography. Forseparation of the optical isomers of amino compounds, addition of chiralcarboxylic or sulfonic acids, such as camphorsulfonic acid, tartaricacid, mandelic acid, or lactic acid can result in formation of thediastereomeric salts.

Alternatively, by method (2), the substrate to be resolved is reactedwith one enantiomer of a chiral compound to form a diastereomeric pair(E. and Wilen, S. “Stereochemistry of Organic Compounds”, John Wiley &Sons, Inc., 1994, p. 322). Diastereomeric compounds can be formed byreacting asymmetric compounds with enantiomerically pure chiralderivatizing reagents, such as menthyl derivatives, followed byseparation of the diastereomers and hydrolysis to yield the pure orenriched enantiomer. A method of determining optical purity involvesmaking chiral esters, such as a menthyl ester, e.g., (−) menthylchloroformate in the presence of base, or Mosher ester,α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob III. J. Org. Chem.(1982) 47:4165), of the racemic mixture, and analyzing the ¹H NMRspectrum for the presence of the two atropisomeric enantiomers ordiastereomers. Stable diastereomers of atropisomeric compounds can beseparated and isolated by normal- and reverse-phase chromatographyfollowing methods for separation of atropisomeric naphthyl-isoquinolines(WO 96/15111). By method (3), a racemic mixture of two enantiomers canbe separated by chromatography using a chiral stationary phase (“ChiralLiquid Chromatography” (1989) W. J. Lough, Ed., Chapman and Hall, NewYork; Okamoto, J. Chromatogr., (1990) 513:375-378). Enriched or purifiedenantiomers can be distinguished by methods used to distinguish otherchiral molecules with asymmetric carbon atoms, such as optical rotationand circular dichroism.

EXAMPLES Example 101a 4-tert-Butylbenzoyl Chloride 101a

A mixture of 4-tert-butylbenzoic acid (1000 g, 5.6 mol) in sulfurousdichloride (1.5 L) was refluxed for 3 hours. Then the mixture wasconcentrated in vacuo, and the crude 101a was used in next step withoutfurther purification.

Example 101b 4-tert-Butyl-N-(2-hydroxy-1,1-dimethyl-ethyl)-benzamide101b

A solution of 101a in DCM (200 mL) was added to a solution of2-amino-2-methyl-1-propanol (1000 g, 10.5 mol) in CH₂Cl₂ (2000 mL)dropwise at 0-10° C. A white precipitate formed 5 minutes after theinitial addition. The resulting slurry was stirred at room temperatureovernight. The solid was removed by filtration and washed with CH₂Cl₂(1000 mL). The filtrate was concentrated on by rotary evaporation togive 101b as a light yellow resin which was used in the next stepwithout further purification.

Example 101c 2-(4-tert-Butyl-phenyl)-4,4-dimethyl-4,5-dihydro-oxazole101c

A mixture of 101b (1000 g, crude) in thionyl chloride (1.5 L) wasrefluxed for 1 hour. The reaction mixture was cooled to room temperatureand poured into 500 mL of stirred Et₂O, during which time a whiteprecipitate formed. The precipitate was collected by filtration andwashed with Et₂O, dissolved in 500 mL of water and neutralized with 25percent NaOH. The yellow aqueous solution was extracted with EtOAC(3×500 mL) and the combined organic phase was washed with 500 mL ofbrine, dried over Na₂SO₄, filtered and concentrated in vacuo to provide101c (530 g, 40.8% over 3 steps) as a white solid. ¹HNMR (300 MHz,CDCl₃) δ 7.87 (d, J=8.4 Hz, 2H), 7.41 (d, J=8.4 Hz, 2H), 4.08 (s, 2H),1.37 (s, 6H), 1.33 (s, 9H).

Example 101d5-tert-Butyl-2-(4,4-dimethyl-4,5-dihydro-oxazol-2-yl)-benzaldehyde 101d

To a solution of 101c (50 g, 0.22 mol) in anhydrous THF (750 mL) wasadded 2.4 M solution of n-butyllithium in hexane (225 mL) at −78° C.under nitrogen atmosphere. The clear amber solution was warmed to −20°C. and stirred for 4 hr. The reaction mixture became red-amber andcloudy. The mixture was re-cooled to −78° C. and stirred rapidly before72 mL of DMF was added drop wise at such a rate that the temperature wascontrolled below −60° C. After the addition, the reaction mixture wasstirred at −78° C. for 15 min then stirred at −20° C. for 1 hr and roomtemperature for 1 hr. The reaction mixture was quenched with 200 mL of0.5 M aqueous KHSO₄. More KHSO₄ solution was added in until the pH wasadjusted to 4-5. The aqueous phase was extracted with EtOAc (3×500 mL)and the combined organic phases was washed with 400 mL of brine anddried over Na₂SO₄, filtered and concentrated in vacuo to give 101d (35g, 62%) as a yellow solid which was used in the next step withoutfurther purification. LCMS, ESI, m/z 260 (M+1)⁺.

Example 101e2-(4-tert-Butyl-2-1,3-dioxinan-2-yl-phenyl)-4,4-dimethyl-4,5-dihydrooxazole101e

A mixture of 101d (60 g, 0.23 mol), pyridinium p-toluenesulfonate (4 g,0.02 mol) and 1,3-propanediol (60 mL) in toluene (500 mL) was heated toreflux overnight and cooled to room temperature upon the completion ofreaction determined by LCMS. The reaction mixture was washed with 200 mLof 50 percent aqueous NaHCO₃, 200 mL of water, and 200 mL of brine. Theorganic layer was dried over Na₂SO₄, filtered and concentrated in vacuoto give a residue which was purified by silica gel chromatography usingEtOAc/petroleum ether=1:5 as eluent to provide 101e (16 g, 21.7%) as aclear yellow gum. LCMS (ESI) 318 (M+H)⁺. ¹HNMR (300 MHz, CDCl₃) δ 7.79(s, 1H), 7.67 (d, J=8.0 Hz, 1H), 7.36 (dd, J=1.6, 8.4 Hz, 1H), 6.32 (s,1H), 4.23 (dd, J=5.2, 11.2 Hz, 2H), 4.06 (s, 2H), 4.04-3.98 (m, 2H),2.27-2.21 (m, 1H), 1.48-1.38 (m, 1H), 1.38 (s, 6H), 1.32 (s, 9H).

Example 101f2-(4-tert-Butyl-2-1,3-dioxinan-2-yl-6-fluoro-phenyl)-4,4-dimethyl-4,5-dihydro-oxazole101f

To a solution of 101e (20 g, 63 mmol) in anhydrous THF (400 mL) wasadded 2.4 M solution of n-butyllithium in hexane (65 mL) at −78° C.under N₂ atmosphere. The clear yellow solution was stirred at −17° C.for 3 hr, when it showed a deep red-orange color. The reaction solutionwas re-cooled to −78° C., stirred rapidly and a solution ofN-fluorobenzenesulfonimide (29 g, 92 mmol) in anhydrous THF (100 mL) wasadded in dropwise over 10 min. The reaction mixture was stirred at −78°C. for 5 min, −20° C. for 30 min, then room temperature for 1 h. Thereaction mixture was poured into 150 mL of 50% aqueous NH₄Cl andextracted with 300 mL of EtOAc. The separated organic phase was washedwith 150 mL of water and 150 mL of brine, dried over Na₂SO₄, filtered,and concentrated in vacuo to give a residue which was purified by silicagel chromatography using EtOAc/CH₂Cl₂=1:5 as eluent to afford 101f (7 g,33%) as yellow solid. LCMS (ESI) m/z 336 (M+H)⁺. ¹HNMR (300 MHz, CDCl₃)δ 7.53 (d, J=1.6 Hz, 1H), 7.08 (dd, J=2.0, 12.0 Hz, 1H), 5.90 (s, 1H),4.24 (dd, J=5.2, 10.8 Hz, 2H), 4.06 (s, 2H), 3.98-3.91 (m, 2H),2.26-2.19 (m, 1H), 1.45-1.42 (m, 1H), 1.42 (s, 6H), 1.30 (s, 9H).

Example 101g 5-tert-Butyl-7-fluoro-3-methoxyisobenzofuran-1(3H)-one 101g

A mixture of 101f (68.8 g, 205.4 mmol), methanol (1340 mL) and 50%aqueous sulfuric acid (881 mL) was stirred at reflux overnight. Thereaction mixture was poured into 400 mL of water, extracted with DCM(3×1000 mL). The combined organic extracts were washed with 400 mL ofbrine, dried over Na₂SO₄, filtered and concentrated to dry to provide101g (43 g) as a off-white solid which was used in the next step withoutfurther purification LCMS (ESI) m/z 225 (M+H)⁺.

Example 101h 6-tert-Butyl-8-fluorophthalazin-1(2H)-one 101h

To a solution of 101g (40 g, 168 mmol) in glacial acetic acid (360 mL)was added hydrazine monohydrate (240 mL) at 0-10° C. under N₂protection. The resulting slurry was stirred under nitrogen at 50° C.for 1.5 hours. The reaction mixture was poured into 300 mL of water withcontinuous stirring. The aqueous phase was extracted with DCM (2×500mL), and the combined organic phase was dried over Na₂SO₄, filtered andconcentrated to give a residue, which was purified by re-crystallizationin DMC and Et₂O to provide 101h (17 g, 37.6% over 2 steps) as anoff-white solid. ¹HNMR (300 MHz, CDCl₃) δ 8.11 (d, J=2.7 Hz, 1H), 7.46(m, 2H), 1.39 (s, 9H). LCMS (ESI) m/z 221 (M+H)⁺.

Example 101i2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-6-chlorobenzaldehyde101i

To a 10 mL round bottom flask were added6-tert-butyl-8-fluoro-2H-phthalazin-1-one 101h (640 mg, 2.9 mmol),2-chloro-6-fluorobenzaldehyde (506 mg, 3.2 mmol), and cesium carbonate(488 mg, 1.5 mmol). The flask was evacuated and backfilled with nitrogenthree times then ethoxytrimethylsilane (684 mg, 5.8 mmol) and DMF (5 mL)were added to the reaction flask. The resulting mixture was heated to60° C. After 4 h of stirring, the solution was allowed to cool down toambient temperature and the reaction was quenched by addition of 2 mL ofH₂O drop-wise. The desired product started to precipitate from the DMFand water mixture. The solid was collected by filtration after coolingdown to 5° C., and washed with DMF/water (2/1, 2 mL, pre-cooled to 6°C.) and H₂O (2 mL). The filter cake was dried under vacuum oven at 65°C. for overnight to afford 519 mg (52%) of 101i as a yellow solid. MS:[M+H]+: 359

Example 101j6-tert-Butyl-2-(3-chloro-2-(hydroxymethyl)phenyl)-8-fluorophthalazin-1(2H)-one101j

2-(6-tert-Butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl)-6-chloro-benzaldehyde101i (519 mg, 1.4 mmol) was dissolved in DCM (2 mL) with stirring atroom temperature, and then 1 mL of iPA was added to the solution. Theresulting solution was cooled to 4° C., and NaBH₄ (27 mg, 0.7 mmol) wasadded in one portion. After 30 min stirring, the reaction was quenchedby adding H₂O (2 mL). The aqueous layer was extracted with CH₂Cl₂ (2×5mL), washed with brine and dried over MgSO₄. The filtrate wasconcentrated to give a residue which was purified by silica gelchromatography eluting with 0-20% EtOAc/CH₂Cl₂ to furnish 101j as awhite solid (385 mg, 72%). MS: [M+H]+: 361

Example 1016-tert-Butyl-8-fluoro-2-(2-(hydroxymethyl)-3-(1-methyl-5-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)phthalazin-1(2H)-one101

To a 10 mL flask were added6-tert-butyl-2-(3-chloro-2-hydroxymethyl-phenyl)-8-fluoro-2H-phthalazin-1-one101j (100 mg, 0.27 mmol),1-methyl-3-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-pyridin-2-one(141 mg, 0.33 mmol), PCy3 (6 mg), Pd(dba)₂ (6 mg) and K₂CO₃ (112 mg, 0.8mmol) in order. The flask was evacuated and backfilled with nitrogen.This sequence was repeated three times. Then, 20 percent aqueous1,4-dioxane was added to the reaction mixture. The resulting mixture washeated to 90° C. for gentle reflux and stirred for 1.5 hr under Nitrogenatmosphere. The reaction mixture was cooled to room temperature andfiltered through a diatomaceous earth filtration agent pad (CELITE®) andconcentrated to afford a residue which was purified by prep-TLC (10%MeOH in CH₂Cl₂) to afford 101 (80 mg, 46%) as a yellow solid. MS:[M+H]⁺624. ¹H NMR (400 MHz, DMSO-d₆) δ 8.53 (d, J=2.0 Hz, 1H), 8.50 (d,J=1.6 Hz, 1H), 8.36 (s, 1H), 7.87 (d, J=3.2 Hz, 2H), 7.73 (d, J=12.8 Hz,1H), 7.51 (t, J=7.6 Hz, 1H), 7.41 (d, J=3.2 Hz, 1H), 7.39 (d, J=3.6 Hz,1H), 7.35 (dd, J=2.8, 9.2 Hz, 1H), 7.28 (d, J=2. Hz, 1H), 7.20 (d, J=9.2Hz, 1H), 4.58 (t, J=4.8 Hz, 1H), 4.36 (s, 2H), 3.58 (s, 3H), 3.04 (m,4H), 2.44 (m, 4H), 2.21 (s, 3H), 1.38 (s, 9H).

Example 102a 2,6-Dibromo-4-fluorobenzaldehyde 102a

A solution of 1,3-dibromo-5-fluoro-2-iodobenzene (50 g, 132 mmol) inanhydrous toluene (300 mL) cooled to −35° C. was added the solution ofisopropylmagnesium chloride (84 mL, 171 mmol, 2.0 M in diethyl ether)over a period of 30 minutes while maintaining the internal temperaturebelow −25° C. A clear brown solution was obtained. Stirring wascontinued for 1.5 h. Then anhydrous DMF (34 mL, 436 mmol) was added overa period of 30 minutes. The temperature of the reaction mixtureincreased to −19° C. The reaction mixture was warmed to 10° C. (roomtemperature) over 1 h and stirred at this temperature for 1.5 h. Thereaction was quenched with saturated aqueous NH₄Cl (100 mL), filteredand evaporated under reduced pressure. The residue was purified bysilica-gel column chromatography (eluting with petroleum ether/ethylacetate: from 50:1 to 20:1) to give 102a (20 g, yield 54%) as a yellowsolid.

Example 102b2-Bromo-6-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-fluorobenzaldehyde102b

To a solution of 102a (767 mg, 2.72 mmol),6-tert-butyl-8-fluorophthalazin-1(2H)-one 101h (300 mg, 1.36 mmol) indioxane (50 mL) was added KOAc (267 mg, 2.72 mmol), CuI (259 mg, 1.36mmol), and 4,7-dimethoxy-1,10-phenanthroline (327 mg, 1.36 mmol). Afterbubbling nitrogen through the resulting solution for 30 min, the mixturewas stirred at 90 degree for 10 h. It was allowed to cool down to roomtemperature and H₂O (100 mL) was added. The aqueous layer was separatedand extracted with ethyl acetate (2×200 mL). The combined organic layerswas washed with brine (100 mL) and dried over sodium sulfate. The dryingagent was removed by filtration and the filtrate was concentrated underreduced pressure. The residue was purified on flash column eluting withPE/EA (15:1) to afford 102b (172 mg, 30%). LCMS: [M+H]⁺ 421. ¹H NMR (500MHz, CDCl₃) δ 10.20 (s, 1H), 8.20 (s, 1H), 7.49-7.51 (m, 3H), 7.25 (m,1H), 1.36 (s, 9H).

Example 102c2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-fluoro-6-(1-methyl-5-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)benzaldehyde102c

A round-bottomed flask was charged with2-bromo-6-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-fluorobenzaldehyde102b (100 mg, 0.24 mmol),1-methyl-3-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one(131 mg, 0.28 mmol), PdCl₂(dppf) (25 mg, 0.03 mmol), K₃PO₄.3H₂O(149 mg,0.56 mmol), THF (10 mL), and H₂O (5 mL). After three cycles ofvacuum/argon flash, the mixture was heated at 70° C. for 6 h. It wasthen filtered and the filtrate was evaporated in vacuo. The residue waspurified on flash column chromatography eluting with 1:3 petroleum/ethylacetate to afford 102c as a yellow solid (98 mg, 60%). LCMS: [M+H]⁺ 682

Example 1026-tert-Butyl-8-fluoro-2-(3-(hydroxymethyl)-4-(1-methyl-5-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-2-yl)phthalazin-1(2H)-one102

A mixture of2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-fluoro-6-(1-methyl-5-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydro-pyridin-3-yl)benzaldehyde102c (98 mg, 0.14 mmol), NaBH₄ (17 mg, 0.43), and CH₃OH (10 mL) wasstirred at 25° C. for 1 h. It was then concentrated at reduce pressureand water (10 mL) was added. The resulting mixture was extracted withCH₂Cl₂ (10 mL×2). The combined CH₂Cl₂ extract was concentrated underreduced pressure and the residue was purified with reverse-phaseprep-HPLC to afford 102 (45 mg, 46%). LCMS: [M+H]⁺ 684. ¹H NMR (500 MHz,DMSO-d₆) δ 8.52-8.56 (m, 2H), 8.40 (s, 1H), 7.88 (d, J=6.0 Hz, 2H),7.74-7.77 (m, 1H), 7.34-7.40 (m, 3H), 7.29 (d, J=3.0 Hz, 1H), 7.22 (d,J=9.5 Hz, 1H), 4.54-4.56 (m, 2H), 4.44-4.47 (m, 2H), 4.32 (s, 2H), 3.58(s, 3H), 3.43 (s, 1H), 3.06-3.08 (m, 4H), 2.36-2.39 (m, 5H), 1.38 (s,9H).

Example 103a 2-Bromo-4-chloronicotinaldehyde 103a

To a solution of 2-bromo-4-chloropyridine (1.6 g, 8.0 mmol) in anhydroustetrahydrofuran (40 mL) cooled at −70° C. was added the solution oflithium diisopropyl-amide (5.0 mL, 10.0 mmol, 2.0 M) over a period of 5minutes and stirred at −70° C. for another 1 h. Anhydrous DMF (1.3 g)was introduced over a period of 3 minutes and the mixture was stirredfor another 30 minutes. It was then quenched with saturated NH₄Cl (30mL) and extracted with ethyl acetate (20 mL×3). The combined organiclayer was dried over anhydrous Mg₂SO₄, filtered, and evaporated underreduced pressure. The residue was purified by silica-gel columnchromatography eluting with petroleum ether/ethyl acetate (20:1) toafford 103a as a yellow solid (900 mg, 48%). ¹H NMR (500 MHz, DMSO-d₆) δ10.21 (s, 1H), 8.52 (d, J=5.5 Hz, 1H), 7.79 (d, J=5.0 Hz, 1H).

Example 103b2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b

Into a solution of 2-bromo-4-chloronicotinaldehyde 103a (446 mg, 2.05mmol), 6-tert-butyl-8-fluorophthalazin-1(2H)-one 101h (300 mg, 1.36mmol) in dioxane (50 mL) was added KAcO (267 mg, 2.72 mmol), CuI(259 mg,1.36 mmol), and 4,7-dimethoxy-1,10-phenanthroline (327 mg, 1.36 mmol).After bubbling nitrogen through the resulting solution for 30 min, themixture was stirred at 90° C. for 10 h. It was allowed to cool down toroom temperature and H₂O (100 mL) was added. The aqueous layer wasseparated and extracted with ethyl acetate (2×200 mL). The combinedorganic layer was washed with brine (100 mL) and dried over sodiumsulfate. The drying agent was removed by filtration and the filtrate wasconcentrated under reduced pressure. The residue was purified on flashcolumn eluting with 10:1 PE/EA to afford 103b (120 mg, 25%). LCMS:[M+H]⁺ 360. ¹H NMR (500 MHz, CDCl₃) δ 10.36 (s, 1H), 8.69 (d, J=5.5,1H), 8.28 (d, J=2.0, 1H), 7.28-7.56 (m, 3H), 1.49 (s, 9H)

Example 103c2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde103c

A mixture of 103b (100 mg, 0.28 mmol),1-methyl-3-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridine-2-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one(131 mg, 0.28 mmol), PdCl₂(dppf) (25 mg, 0.03 mmol), and K₃PO₄.3H₂O (149mg, 0.56 mmol) was suspended in THF (10 mL)/H₂O (5 mL). After threecycles of vacuum/argon flash, the mixture was heated at 70° C. for 6 h.It was then filtered and the filtrate was evaporated in vacuo. Theresidue was purified on flash column chromatography eluting with 1:3petroleum/ethyl acetate to afford 103c as a yellow solid (150 mg, 60%).LCMS: [M+H]⁺ 665

Example 1036-tert-butyl-8-fluoro-2-(3-(hydroxymethyl)-4-(1-methyl-5-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-2-yl)-phthalazin-1(2H)-one103

A mixture of 103c (120 mg, 0.18 mmol), NaBH₄ (21 mg, 0.54), and CH₃OH(10 mL) was stirred at 25° C. for 1 h. It was then concentrated underreduced pressure and water (10 mL) was added. The resulting mixture wasextracted with CH₂Cl₂ (10 mL×2). The combined CH₂Cl₂ extract wasconcentrated under reduced pressure and the residue was purified withreverse-phase prep-HPLC to afford 103 (45 mg, 38%). LCMS: [M+H]⁺ 667. ¹HNMR (500 MHz, CDCl₃) δ 8.66-8.68 (m, 2H), 8.35 (s, 1H), 7.96 (d, J=3.0,1H), 7.81 (s, 1H), 7.65 (d, J=2.5, 1H), 7.55-7.59 (m, 3H), 6.83 (d,J=8.5, 1H), 4.71-4.74 (m, 4H), 4.51-4.52 (m, 2H), 4.07-4.08 (m, 1H),3.73 (s, 3H), 3.56 (s, 1H), 3.20-3.22 (m, 4H), 2.56-2.58 (m, 4H), 1.59(s, 9H)

Example 104a(S)-2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-fluoro-6-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)benzaldehyde104a

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with2-bromo-6-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-fluorobenzaldehyde102b (100 mg, 0.24 mmol),(S)-1-methyl-3-(5-(2-methyl-4-(oxe-tan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one105f (115 mg, 0.24 mmol), PdCl₂(dppf) (22 mg, 0.03 mmol), K₃PO₄ (102 mg,0.48 mmol), sodium acetate (39 mg, 0.48 mmol), THF (15 mL), and water (5mL). After three cycles of vacuum/argon flush, the mixture was heated at100° C. for 2 h. It was then filtered and the filtrate was evaporatedunder reduced pressure. The residue was purified by silica-gel columnchromatography eluting with 40:1 dichloromethane/methanol to afford 104aas a yellow solid (82 mg, 49%). MS: [M+H]⁺ 696.3

Example 104(S)-6-tert-Butyl-8-fluoro-2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)phthalazin-1(2H)-one104

A mixture of(S)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-fluoro-6-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)benzaldehyde104a (80 mg, 0.12 mmol), NaBH₄ (14 mg, 0.36), and methanol (20 mL) wasstirred at 25° C. for 1 h. Then the reaction mixture was quenched withwater (10 mL) and concentrated under reduced pressure. The residue wasextracted with dichloromethane (2×30 mL) and the combineddichloromethane extract was concentrated under reduced pressure. Theresidue was purified by reverse-phase prep-HPLC to afford 104 (46 mg,55%) as a yellow solid. MS: [M+H]⁺ 698.3. ¹H NMR (500 MHz, DMSO-d₆) δ8.54 (d, J=2.0 Hz, 1H), 8.52 (d, J=2.0 Hz, 1H), 8.42 (s, 1H), 7.88 (s,1H), 7.86 (d, J=3.0 Hz, 1H), 7.77-7.74 (m, 1H), 7.40-7.34 (m, 3H), 7.30(dd, J=2.5, 9.5 Hz, 1H), 7.23 (d, J=9.0 Hz, 1H), 4.57-4.54 (m, 2H),4.49-4.45 (m, 1H), 4.43-4.40 (m, 1H), 4.34-4.29 (m, 2H), 3.70-3.65 (m,1H), 3.58 (s, 3H), 3.40-3.38 (m, 2H), 3.12-3.06 (m, 1H), 2.97-2.91 (m,1H), 2.36-2.29 (m, 3H), 2.19-2.15 (m, 1H), 1.38 (s, 9H), 0.93 (d, J=6.5Hz, 3H),

Example 105a (3S)-tert-Butyl3-Methyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 105a

Into a solution of 5-bromo-2-nitropyridine (30 g, 148 mmol) in DMSO (350mL) were added K₂CO₃ (14 g, 104 mmol) and(3S)-tert-butyl-3-methylpiperazine-1-carboxylate (10.0 g, 50 mmol). Themixture was stirred at 65° C. overnight and then after cooling to roomtemperature, poured into water (700 mL). The solid precipitate wascollected and dried under vacuum. It was further purified on a flashcolumn eluting with 20:1 petroleum ether/ethyl acetate and then withCH₂Cl₂ to give 105a as a yellow solid (8.05 g, 50%). LCMS: [M+H]⁺ 323

Example 105b (3S)-tert-Butyl4-(6-Aminopyridin-3-yl)-3-methylpiperazine-1-carboxylate 105b

A 500-mL bottle was purged with nitrogen and charged with 105a (5.8 g,18 mmol), 10% palladium on carbon (50% wet, 2.0 g) and ethanol (200 mL).It was evacuated, charged with hydrogen gas, and stirred for 16 h atroom temperature. The hydrogen was then removed in vacuo and replacedwith nitrogen. The catalyst was removed by filtration through a pad ofCELITE® and the filtrate concentrated under reduced pressure to afford105b as a brown solid (4.9 g, 96%). LCMS: [M+H]⁺ 293

Example 105c(3S)-tert-Butyl-4-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridine-3-yl)-3-methylpiperazine-1-carboxylate105c

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and reflux condenser was charged with 1,4-dioxane (50 mL), 105b(4.0 g, 13.7 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (5.5 g, 20.8mmol), and cesium carbonate (11 g, 35 mmol). After bubbling nitrogenthrough the resulting mixture for 30 minutes, XantPhos (272 mg, 0.47mmol) and tris(dibenzylideneacetone)dipalladium(0) (430 mg, 0.47 mmol)were added, and the reaction mixture was heated at reflux for 3 h. Afterthis time the reaction was cooled to room temperature, partitionedbetween ethyl acetate (300 mL) and water (300 mL), and filtered. Theaqueous layer was separated and extracted with ethyl acetate (150 mL×2).The organic layers were combined, washed with brine (150 mL), and driedover sodium sulfate. The drying agent was removed by filtration and thefiltrate was concentrated under reduced pressure. The residue waspurified on flash column eluting with 50:1 CH₂Cl₂/methanol to afford105c as a yellow solid (5.4 g, 83%). LCMS: [M+H]⁺ 478

Example 105d(3S)-5-Bromo-1-methyl-3-(5-(2-methylpiperazin-1-yl)pyridin-2-ylamino)pyridine-2(1H)-one105d

A mixture of 105c (3.1 g, 6.5 mmol) and 4.0 M HCl/dioxane (10 mL) wasstirred for 5 h at room temperature. It was then concentrated in vacuo.The residue was basified with aqueous 1.0M NaOH and extracted twice withCH₂Cl₂. The combined organic layers were washed with water andconcentrated under reduced pressure to give 105d as a yellow solid (2.3g, 95%). LCMS: [M+H]⁺ 380.

Example 105e(3S)-5-Bromo-1-methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazine-1-yl)pyridine-2-ylamino)pyridin-2(1H)-one105e

A mixture of 105d (2.35 g, 6.2 mmol) and oxetan-3-one (0.49 g, 6.8 mmol)NaBH₃CN (4.75 g, 22.5 mmol), and zinc chloride (3 g, 22.7 mmol) inmethanol (125 mL) was stirred for 5 hours at 50° C. The mixture wasadded to water and extracted with CH₂Cl₂ three times. The organic layerswere concentrated under reduced pressure. The residue was purified bycolumn chromatography eluting with 25:1 CH₂Cl₂/methanol to give 105e asa yellow solid (2.6 g, 98%). LCMS: [M+H]⁺ 434.

Example 105f(3S)-1-methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazine-1-yl)pyridin-2-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one105f

A 100 mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 105e (1.0 g, 1.0 eq.,2.3 mmol), Pin₂B₂ (1.46 g, 2.50 eq., 5.75 mmol), Pd₂(dba)₃ (105 mg, 0.05eq., 0.125 mmol), X-Phos (93 mg, 0.1 eq., 0.23 mmol), KOAc (676 mg, 3.0eq., 6.9 mmol), and dioxane (50 mL). After three cycles of vacuum/argonflush, the mixture was heated at 90° C. for 4 h. It was then cooled toroom temperature and filtered. The filtrate was concentrated underreduced pressure and the resulting residue was washed with 3:1 petroleumether/ethyl acetate (80 mL) to afford 105f as yellow solid (1.0 g, 90%).MS: [M+H]⁺ 482.

Example 105g(S)-2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde105g

A round-bottomed flask was charged with2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (100 mg, 0.28 mmol), 105f (135 mg, 0.28 mmol), PdCl₂(dppf) (25 mg,0.03 mmol), K₃PO₄.3H₂O(149 mg, 0.56 mmol), THF (10 mL), and H₂O (5 mL).After three cycles of vacuum/argon flush, the mixture was heated at 70°C. for 6 h. It was then filtered and the filtrate was evaporated invacuo. The residue was purified on flash column chromatography elutingwith 1:3 petroleum/ethyl acetate to afford 105g as a yellow solid (113mg, 60%). LCMS: [M+H]⁺ 679

Example 105(S)-6-tert-butyl-8-fluoro-2-(3-(hydroxymethyl)-4-(1-methyl-5-(3-methyl-5-(4-(oxetan-3-yl)piperazine-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-2-yl)phthalazin-1(2H)-one105

A mixture of 105g (100 mg, 0.15 mmol), NaBH₄ (17 mg, 0.45), and CH₃OH(10 mL) was stirred at 25° C. for 1 h. The mixture was extracted withCH₂Cl₂ (10 mL×2). The combined CH₂Cl₂ extracts were concentrated underreduced pressure. The residue was purified by reverse-phase prep-HPLC toafford 105 (65 mg, 65%). LCMS: [M+H]⁺ 681. ¹H NMR (500 MHz, DMSO-d6) δ8.62 (d, J=2.0, 1H), 8.53-8.57 (m, 1H), 8.42 (s, 2H), 7.90 (d, J=1.5,1H), 7.85 (d, J=2.0, 1H), 7.76-7.79 (m, 1H), 7.52 (d, J=5.0, 1H), 7.46(d, J=2.0, 1H), 7.36-7.38 (m, 1H), 7.24-7.26 (m, 1H), 4.54-4.57 (m, 2H),4.46-4.48 (m, 1H), 4.40-4.41 (m, 3H), 3.68-3.70 (m, 1H), 3.57 (s, 3H),3.37-3.40 (m, 1H), 3.09-3.11 (m, 1H), 2.93-2.95 (m, 1H), 2.52-2.54 (m,2H), 2.32-2.36 (m, 2H), 2.18 (s, 1H), 1.39 (s, 9H), 0.93 (d, J=6.0, 3H)

Example 106a(R)-1-Methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazine-1-yl)pyridin-2-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one106a

To a solution of(R)-5-bromo-1-methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazine-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one(2.0 g, 4.60 mmol),4,4,4′,4′,5,5,5′,5′-octa-methyl-2,2′-bi(1,3,2-dioxaborolane) (3.50 g,13.80 mmol) in dioxane (50 mL) was added PdCl₂(dppf) (377.10 mg, 0.46mmol) and KOAc (2.70 g, 27.80 mmol). See FIG. 6. The mixture was stirredat 100° C. for 12 h under argon. The mixture was filtered and thefiltrate was evaporated in vacuo. The residue was purified by columnchromatography eluting with CH₂Cl₂/methanol (15:1) to give 106a (1.10 g,49%) as a brown solid. MS: [M+H]⁺ 482.3

Example 106b(R)-2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazine-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde106b

A mixture of 106a (260.0 mg, 0.56 mmol),2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (200.0 mg, 0.56 mmol), PdCl₂(dppf) (50.0 mg, 0.056 mmol), NaOAc(90.0 mg, 1.1 mmol), K₃PO₄ (300 mg, 1.1 mmol) in acetonitrile (30 mL)was heated at 100° C. for 3 h. The solvent was evaporated in vacuo. Theresidue was purified by flash column chromatography eluting withCH₂Cl₂/methanol (10:1) to afford 106b (120 mg, 34%) as a brown solid.LCMS: [M+H]⁺ 679.3

Example 106(R)-6-tert-Butyl-8-fluoro-2-(3-(hydroxymethyl)-4-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazine-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-2-yl)phthalazin-1(2H)-one106

A solution of 106b (90.0 mg, 0.13 mmol) in MeOH (10 mL) was added NaBH₄(15.0 mg, 0.39 mmol). The mixture was stirred at 20° C. for 2 h. It wasthen evaporated and the residue was purified by reverse-phase prep-HPLCto afford 106 (7.3 mg, 8%) as a white solid. LCMS: (M+H)⁺ 681.4. ¹H NMR(500 MHz, DMSO) δ 8.63 (d, J=2.5, 1H), 8.57 (d, J=5.0, 1H), 8.54 (d,J=2.0, 1H), 8.47 (s, 1H), 7.91 (s, 1H), 7.86 (d, J=3.0, 1H), 7.78 (d,J=12.5, 1H), 7.53 (d, J=5.0, 1H), 7.47 (d, J=2.0, 1H), 7.36-7.39 (m,1H), 7.25 (d, J=9.5, 1H), 4.90 (s, 1H), 4.54-4.58 (m, 2H), 4.47 (t,J=6.0, 1H), 4.39-4.43 (m, 3H), 3.69 (s, 1H), 3.61 (s, 3H), 3.37-3.42 (m,1H), 3.09-3.11 (m, 1H), 2.95 (t, J=9.0, 1H), 2.54-2.56 (m, 1H),2.30-2.37 (m, 2H), 2.19 (t, J=8.0, 1H), 1.39 (s, 9H), 0.94-0.93 (d,J=6.5, 3H)

Example 107a(R)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-fluoro-6-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydro-pyridin-3-yl)benzaldehyde107a

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with(R)-1-methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one(173 mg, 1.0 eq., 0.36 mmol),2-bromo-6-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-fluorobenzaldehyde101i (152 mg, 1.0 eq., 0.36 mmol), K₃PO₄ (152 mg, 2.0 eq., 0.72 mmol),PdCl₂(dppf) (26 mg, 0.1 eq., 0.036 mmol), NaOAc (59 mg, 2.0 eq., 0.72mmol), CH₃CN (20 mL), and H₂O (0.8 mL). After three cycles ofvacuum/argon flush, the mixture was heated at 80° C. for 2 h. It wasthen cooled to room temperature and filtered. The filtrate wasconcentrated under reduced pressure and the resulting residue waspurified by silica-gel column chromatography eluting with 40:1 DCM/EtOHto afford 107a as yellow solid (77 mg, 31%). MS: [M+H]⁺ 696.3.

Example 107(R)-6-tert-Butyl-8-fluoro-2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)phthalazin-1(2H)-one107

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with 107a (77 mg, 1.0 eq., 0.11 mmol), NaBH₄ (21 mg,5.0 eq., 0.55 mmol), and MeOH (10 mL). The mixture was stirred at roomtemperature for 1 h. It was then filtered and the filtrate wasconcentrated. The residue was purified by reverse-phase prep-HPLC toafford 107 (38 mg, 49%). MS: [M+H]⁺ 698.3. ¹H NMR (500 MHz, CDCl₃) δ8.59 (d, J=2.0 Hz, 1H), 8.29 (d, J=2.5 Hz, 1H), 7.98 (d, J=2.5 Hz, 1H),7.81 (s, 1H), 7.56-7.54 (m, 2H), 7.40 (s, 1H), 7.31-7.27 (m, 2H),7.11-7.10 (m, 1H), 6.81 (d, J=9.0 Hz, 1H), 4.71-4.62 (m, 4H), 4.35 (s,2H), 3.73-3.70 (m, 4H), 3.53-3.46 (m, 2H), 3.08 (t, J=5.0 Hz, 2H),2.56-2.46 (m, 3H), 2.22-2.18 (m, 1H), 1.43 (s, 9H), 0.98 (d, J=6.5 Hz,3H).

Example 108a2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-6-(5-(5-((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluorobenzaldehyde108a

A 25-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with2-bromo-6-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-fluorobenzaldehyde102b (84 mg, 0.20 mmol),3-(5-((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one(99 mg, 0.20 mmol), Pd(dppf)Cl₂ (15 mg, 0.02 mmol), K₃PO₄ (127 mg, 0.6mmol), sodium acetate (49 mg, 0.6 mmol), acetonitrile (5 mL), and water(0.5 mL). After three cycles of vacuum/argon flush, the mixture washeated at reflux for 2 h. It was then cooled to room temperature andfiltered. The filtrate was concentrated under reduced pressure and theresulting residue was purified by silica-gel column chromatographyeluting with 30:1 dichloromethane/methanol to 108a as a white solid (54mg, 38%). MS: [M+H]⁺ 710.3

Example 1086-tert-Butyl-2-(3-(5-(5-((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-8-fluorophthalazin-1(2H)-one108

At 0° C., to a solution of2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-6-(5-(5-((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluorobenzaldehyde108a (54 mg, 0.076 mmol) in methanol (5 mL) was added sodium borohydride(9.0 mg, 0.23 mmol). The mixture was stirred for 60 minutes. It was thenquenched with water (1.0 mL) and concentrated under reduced pressure.The residue was purified by reverse-phase prep-HPLC to afford 108 (12mg, 22%). MS: [M+H]⁺ 712.3. ¹H NMR (500 MHz, CDCl₃) δ 8.63 (d, J=2.5 Hz,1H), 8.31 (d, J=2.0 Hz, 1H), 8.05 (d, J=2.5 Hz, 1H), 7.87 (s, 1H),7.58-7.55 (m, 2H), 7.44 (d, J=2.0 Hz, 1H), 7.36 (s, 1H), 7.30 (d, J=2.0Hz, 1H), 7.12 (dd, J=2.5, 8.5 Hz, 1H), 6.82 (d, J=8.5 Hz, 1H), 4.76-4.73(m, 2H), 4.68-4.61 (m, 2H), 4.38-4.36 (m, 2H), 3.74-3.72 (m, 2H), 3.72(s, 3H), 3.22-3.20 (m, 1H), 2.94-2.92 (m, 1H), 2.74-2.72 (m, 2H),2.50-2.48 (m, 1H), 1.98-1.96 (m, 1H), 1.45 (s, 9H), 0.93-0.91 (m, 6H).

Example 109a3-Bromo-5-(6-tert-butyl-8-fluoro-1-oxo-1,2-dihydrophthalazin-2-yl)pyridine-4-carbaldehyde109a

A sealed tube equipped with a magnetic stirrer was charged with6-tert-butyl-8-fluoro-1,2-dihydrophthalazin-1-one 101h (220 mg, 1.0mmol), 3,5-dibromopyridine-4-carbaldehyde (530 mg, 2.0 mmol), CuI (190mg, 1.0 mmol), 4,7-dimethoxy-1,10-phenanthroline (244 mg, 1.0 mmol),Cs₂CO₃ (652 mg, 2.0 mmol) and dioxane (8 mL). After three cycles ofvacuum/argon flush, the mixture was heated at 110° C. for 5 h. It wasthen filtered and the filtrate was evaporated in vacuo. The residue waspurified by silica gel column chromatography eluting with ethylacetate/petroleum ether (1:3, V/V) to afford 109a (118 mg, 29.3%) as asolid. LCMS: [M+H]⁺ 406

Example 109b3-(6-tert-Butyl-8-fluoro-1-oxo-1,2-dihydrophthalazin-2-yl)-5-[1-methyl-5-({5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydro-pyridin-3-yl]pyridine-4-carbaldehyde109b

A 25 mL single-neck round-bottomed flask equipped with a magneticstirrer and reflux condenser was charged with 109a (118 mg, 0.29 mmol),1-methyl-3-({5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-5-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2-dihydropyridin-2-one105f (140 mg, 0.29 mmol), Pd(dppf)Cl₂ (24.8 mg, 0.03 mmol), KOAc (58.9mg, 0.60 mmol), K₃PO₄.3H₂O (159.8 mg, 0.60 mmol), acetonitrile (6 mL)and water (3 d). After three cycles of vacuum/argon flush, the mixturewas heated at 110° C. for 2 h. The reaction mixture was then cooled toroom temperature, filtered and the filtrate was evaporated in vacuo. Theresidue was purified by prep-TLC developing withdichloromethane/methanol (20:1, UV) to afford 109b (95 mg, 36%) as a redsolid. MS: [M+H]⁺ 679

Example 109(S)-6-tert-butyl-8-fluoro-2-(4-(hydroxymethyl)-5-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)phthalazin-1(2H)-one109

To a suspension of 109b (95 mg, 0.106 mmol) at 0° C. in methanol (5 mL)was added sodium borohydride (24 mg, 0.636 mmol) and the mixture wasstirred for 30 minutes. Then the reaction mixture was quenched withwater (1.0 mL) and concentrated. The residue was purified byreverse-phase prep-HPLC to afford 109 (13.5 mg, 18.7%). LCMS: [M+H]⁺681.

¹H NMR (500 MHz, CDCl₃) δ 8.76 (s, 1H), 8.65 (d, J=2.0, 1H), 8.61 (s,1H), 8.32 (d, J=2.5, 1H), 7.96 (d, J=3.0, 1H), 7.82 (s, 1H), 7.56-7.58(m, 2H), 7.40 (d, J=2.0, 1H), 7.28-7.31 (m, 1H), 6.81 (d, J=9.0, 1H),4.61-4.70 (m, 4H), 4.43 (s, 2H), 3.95 (s, 1H), 3.72 (s, 3H), 3.45-3.53(m, 2H), 3.07 (t, J=5.25, 2H), 2.43-2.56 (m, 3H), 2.18-2.22 (m, 1H),1.43 (s, 9H), 0.99 (d, J=6.5, 3H)

Example 110a2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(5-((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde110a

A 100-mL single-neck round-bottomed flask was charged with2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (216 mg, 0.6 mmol),3-(5-((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-5-(4,4,5-trimethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one(360 mg, 0.72 mmol), Pd(dppf)Cl₂ (30 mg, 0.03 mmol), K₃PO₄ (270 mg, 1.2mmol), and NaOAc.3H₂O (180 mg, 1.2 mmol) in CH₃CN (80 mL). The systemwas evacuated and refilled with N₂. The reaction mixture was heated at100° C. for 2 h. It was then cooled to room temperature and filtered.The filtrate was concentrated under reduced pressure and the resultingresidue was purified by flash column chromatography eluting with 25:1 ofCH₂Cl₂/MeOH to afford 110a (160 mg, 42%) as a yellow brown solid. MS:[M+H]⁺ 693.3.

Example 1106-tert-Butyl-2-(4-(5-(5-((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-3-(hydroxymethyl)pyridin-2-yl)-8-fluorophthalazin-1(2H)-one110

A mixture of 110a (100 mg, 0.15 mmol) and NaBH₄ (20 mg, 0.45 mmol) inMeOH (30 mL) was stirred at 30° C. for 2 h. The mixture was quenchedwith water and extracted with EtOAc (10 mL×3). The combined EtOAcextract was concentrated under reduced pressure and the residue waspurified by reverse-phase prep-HPLC to afford 110 (80 mg, 80%). MS:[M+H]⁺ 695.3. ¹H NMR (500 MHz, CDCl₃) δ 8.72 (d, J=2, 1H), 8.67 (d, J=5,1H), 8.35 (d, J=2.5, 1H), 8.05 (d, J=3, 1H), 7.88 (s, 1H), 7.68 (d,J=2.5, 1H), 7.58-7.55 (m, 3H), 7.38-7.36 (m, 1H), 6.82 (d, J=8.5, 1H),4.78-4.71 (m, 2H), 4.67-4.61 (m, 2H), 4.50 (s, 2H), 4.07 (t, J=6, 1H),3.78-3.74 (m, 4H), 3.22-3.20 (m, 1H), 2.92 (d, J=3, 1H), 2.77-2.71 (m,2H), 2.51-2.48 (m, 1H), 1.20-1.95 (m, 1H), 1.45 (s, 9H), 0.93-0.90 (m,6H)

Example 111a (S)-tert-Butyl3-Ethyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 111a

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (50 mL),5-bromo-2-nitropyridine (2.02 g, 10 mmol), (S)-tert-butyl3-ethylpiperazine-1-carboxylate (2.14 g, 10.0 mmol), Pd₂(dba)₃ (458 mg,0.50 mmol), XantPhos (576 mg, 1.0 mmol), and cesium carbonate (6.52 g,20 mmol). After three cycles of vacuum/argon flush, the mixture washeated at 100° C. overnight. After this time the reaction was cooled toroom temperature. It was then filtered and the filtrate was evaporatedunder reduced pressure. The residue was purified by silica-gel columnchromatography eluting with 3:1 petroleum ether/ethyl acetate to afford111a (700 mg, 22%) as a yellow solid. MS: [M+H]⁺ 336

Example 111b (S)-tert-Butyl4-(6-Aminopyridin-3-yl)-3-ethylpiperazine-1-carboxylate 111b

A 100-mL single-neck round-bottomed flask was purged with nitrogen andcharged with 111a (0.7 g, 2.08 mmol), 10% palladium on carbon (50% wet,208 mg), and methanol (40 mL). The mixture was evacuated, charged withhydrogen gas, and stirred at room temperature for 6 h. The hydrogen wasthen evacuated and nitrogen was charged into the flask. The catalyst wasremoved by filtration through a pad of CELITE® and the filtrate wasconcentrated under reduced pressure to afford 111b (568 mg, 89%). MS:[M+H]⁺ 306

Example 111c (S)-tert-Butyl4-(6-(5-Bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yl)-3-ethylpiperazine-1-carboxylate111c

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (50 mL),111b (568 mg, 1.86 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (498 mg,1.86 mmol), Pd₂(dba)₃ (85 mg, 0.093 mmol), XantPhos (107 mg, 0.186mmol), and cesium carbonate (1.198 g, 3.72 mmol). After three cycles ofvacuum/argon flush, the mixture was heated at 100° C. for 6 h. It wasthen filtered and the filtrate was evaporated under reduced pressure.The residue was purified by silica-gel column chromatography elutingwith 100:1 dichloromethane/methanol to afford 111c (502 mg, 55%) as ayellow solid. MS: [M+H]⁺ 492.

Example 111d(S)-5-Bromo-3-(5-(2-ethylpiperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one111d

A mixture of 111c (502 mg, 1.02 mmol), dichloromethane (2 mL), and 4.0 MHCl/dioxane (4 mL) was stirred at room temperature for 5 h. It was thenconcentrated under reduced pressure to afford crude 111d as a yellowsolid (263 mg, 66%), which was used in the next step without furtherpurification. MS: [M+H]⁺ 392.

Example 111e(S)-5-Bromo-3-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one111e

A mixture of 111d (263 mg, 0.67 mmol), oxetan-3-one (96 mg, 1.34 mmol),NaBH₃CN 104 mg, 1.68 mmol), and zinc chloride (227 mg, 1.68 mmol) inmethanol (10 mL) was stirred at 50° C. for 5 hours. water (10 mL) wasthen added to the reaction. The resulting mixture was concentrated underreduced pressure. The residue was extracted with dichloromethane threetimes. The combined organic layer was concentrated under reducedpressure and the residue was purified by silica-gel columnchromatography eluting with 50:1 dichloromethane/methanol to afford 111e(203 mg, 68%). MS: [M+H]⁺ 448.

Example 111f(S)-3-(5-(2-Ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one111f

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 111e (3219 mg, 7.20mmol), Pin₂B₂ (9072 mg, 36.0 mmol), Pd₂(dba)₃ (329 mg, 0.36 mmol),X-phos (302 mg, 0.72 mmol), potassium acetate (2117 mg, 21.6 mmol), anddioxane (50 mL). After three cycles of vacuum/argon flush, the mixturewas heated at 60° C. for 16 h. It was then cooled to room temperatureand filtered. The filtrate was concentrated under reduced pressure andthe resulting residue was washed with 8:1 petroleum ether/ethyl acetate(80 mL) to afford 111f as a yellow solid (3.0 g, 84%).

Example 111g(S)-2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-6-(5-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluorobenzaldehyde111g

A 25-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with2-bromo-6-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-fluorobenzaldehyde102h (168 mg, 0.40 mmol), 111f (198 mg, 0.40 mmol), Pd(dppf)Cl₂ (15 mg,0.02 mmol), K₃PO₄ (170 mg, 0.8 mmol), sodium acetate (66 mg, 0.8 mmol),acetonitrile (5 mL), and water (0.8 mL). After three cycles ofvacuum/argon flush, the mixture was heated at reflux for 2 h. It wasthen cooled to room temperature and filtered. The filtrate wasconcentrated under reduced pressure and the resulting residue waspurified by silica-gel column chromatography eluting with 30:1dichloromethane/methanol to afford 111g as a white solid (100 mg, 35%).MS: [M+H]⁺ 710.3

Example 111(S)-6-tert-Butyl-2-(3-(5-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl-amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-8-fluorophthalazin-1(2H)-one111

To a solution of 111g (100 mg, 0.14 mmol) at 0° C. in methanol (5 mL)was added sodium borohydride (16.0 mg, 0.42 mmol). The mixture wasstirred for 60 minutes. It was then quenched with water (1.0 mL) andconcentrated. The residue was purified by reverse-phase prep-HPLC toafford 111 (32 mg, 32%). MS: [M+H]⁺ 712.3. ¹H NMR (500 MHz, CDCl₃) δ8.57 (d, J=2.0 Hz, 1H), 8.30 (d, J=2.0 Hz, 1H), 7.94 (s, 1H), 7.80 (s,1H), 7.58 (s, 2H), 7.41 (d, J=2.0 Hz, 1H), 7.31-7.28 (m, 2H), 7.12 (dd,J=2.5, 8.5 Hz, 1H), 6.82 (d, J=9.0 Hz, 1H), 4.73-4.70 (m, 4H), 4.37 (s,2H), 3.74-3.72 (m, 1H), 3.71 (s, 3H), 3.56-3.54 (m, 1H), 3.34-3.32 (m,1H), 3.15-3.13 (m, 2H), 2.58-2.36 (m, 4H), 1.45 (s, 9H), 1.44-1.43 (m,2H), 0.83 (t, J=7.5 Hz, 3H).

Example 112a(S)-2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde112a

A 50-mL round bottom flask was charged with2-(6-tert-butyl-8-fluoro-1-oxo-phthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (150 mg, 0.43 mmol),(S)-3-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one(206 mg, 0.43 mmol), PdCl₂(dppf) (33 mg, 0.04 mmol), K₃PO₄ (202 mg, 0.86mmol), NaOAc (71 mg, 0.86 mmol), and CH₃CN (10 mL), H₂O (2 mL). Afterthree cycles of vacuum/argon flush, the mixture was heated at 100° C.for 3 h. It was then filtered and the filtrate was evaporated in vacuo.The residue was purified by flash column chromatography eluting with 1:3petroleum/ethyl acetate to afford 112a as a yellow solid (146 mg, 49%).LCMS: [M+H]⁺ 693

Example 112(S)-6-tert-Butyl-2-(4-(5-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-3-(hydroxymethyl)pyridin-2-yl)-8-fluorophthalazin-1(2H)-one112

A mixture of 112a (140 mg, 0.20 mmol), NaBH₄ (21 mg, 0.60) and CH₃OH (8mL) was stirred at 25° C. for 1 h. Then the reaction mixture wasquenched with water (10 mL), the mixture was extracted with CH₂Cl₂ (15mL×2). The combined CH₂Cl₂ extract was concentrated under reducedpressure. The residue was purified with reverse-phase prep-HPLC toafford 112 (70 mg, 50%). LCMS: [M+H]⁺ 695. ¹H NMR (500 MHz, DMSO) δ8.50-8.60 (m, 2H), 8.44 (s, 1H), 7.90 (d, J=1.5, 1H), 7.82 (d, J=3.0,1H), 7.75-7.78 (m, 1H), 7.51-7.53 (m, 1H), 7.45 (d, J=2.5, 1H),7.33-7.35 (m, 1H), 7.22-7.24 (m, 1H), 4.47-4.59 (m, 2H), 4.36-4.45 (m,4H), 3.60 (s, 3H), 3.46-3.50 (m, 1H), 3.38-3.41 (m, 3H), 3.14-3.17 (m,1H), 2.96-3.00 (m, 1H), 2.60-2.64 (m, 1H), 2.50-2.55 (m, 1H), 2.14-2.17(m, 1H), 2.06-2.10 (m, 1H), 1.66-1.69 (m, 1H), 1.39 (s, 9H), 1.21-1.28(m, 1H), 0.77-0.80 (m, 3H)

Example 113a 5-Bromo-1-methyl-3-(pyrimidin-4-ylamino)pyridin-2(1H)-one113a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and nitrogen inlet was charged with3,5-dibromo-1-methylpyridin-2(1H)-one (2.00 g, 21.0 mmol),2-aminopyrimidine (5.61 g, 21.0 mmol), cesium carbonate (13.7 g, 42.1mmol), DMF (5 mL) and 1,4-dioxane (70 mL). After bubbling nitrogenthrough the resulting suspension for 30 min, Xantphos (1.10 g, 1.89mmol) and tris(dibenzylideneacetone)dipalladium(0) (963 mg, 1.05 mmol)were added. A reflux condenser was attached to the flask, and thereaction mixture was heated at 100° C. for 4 h. After this time, themixture was cooled to room temperature and diluted with 90:10 methylenechloride/methanol (150 mL) and water (100 mL), and the layers wereseparated. The aqueous layer was extracted with 90:10 methylenechloride/methanol (50 mL), and the combined organic layers were washedwith brine and dried over sodium sulfate. The drying agent was removedby filtration. The filtrate was concentrated under reduced pressure, andthe resulting residue was purified by flush column chromatography(silica, 90:10 methylene chloride/methanol) to afford 113a in 58% yield(3.42 g) as an amorphous light green solid: mp 217-219° C.; ¹H NMR (500MHz, CDCl₃) δ 9.29 (s, 1H), 8.77 (s, 1H), 8.72 (d, J=2.5 Hz, 1H), 8.36(d, J=6.0 Hz, 1H), 7.69 (d, J=2.5 Hz, 1H), 7.37 (dd, J=5.5, 1.0 Hz, 1H),3.53 (s, 3H); MS (ESI+) m/z 281.0 (M+H).

Example 113b1-Methyl-3-(pyrimidin-4-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one113b

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer and a condenser was charged with 113a (4.0 g, 14 mmol), X-phos(400 mg, 0.7 mmol), Pd2(dba)3 (635 mg, 0.7 mmol), potassium acetate (7.3mg, 28 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (10.6 g, 42mmol), and 1,4-dioxane (100 mL). After three cycles of vacuum/argonflush, the reaction mixture was heated at 60° C. for 8 h. It was thencooled to room temperature and filtered. The filtrate was concentratedunder reduced pressure and the resulting residue was purified by flushcolumn chromatography eluting with 5:1 petroleum ether/ethyl acetate toafford 113b as a pale yellow solid (3.8 mg, 82%). MS: [M+H]⁺ 329.5.

Example 113c2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-1,6-dihydropyridin-3-yl)nicotinaldehyde113c

A 100-mL round-bottomed flask equipped with a reflux condenser wascharged with2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (110 mg, 0.31 mmol), 113b (100 mg, 0.31 mmol), PdCl₂(dppf) (25 mg,0.030 mmol), K₃PO₄ (241 mg, 0.93 mmol), NaAcO (76 mg, 0.93 mmol),acetonitrile (15 mL), and water (0.5 mL). After three cycles ofvacuum/argon flush, the mixture was heated at 100° C. for 3 h. It wasthen filtered and the filtrate was evaporated under reduced pressure.The residue was purified with silica-gel column chromatography elutingwith 1:20 methanol/dichloromethane to afford 113c as a red solid (80 mg,51%). MS-ESI: [M+H]⁺ 526.3

Example 1136-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-6-oxo-5-(pyrimidin-4-ylamino)-3-pyridyl]-2-pyridyl]phthalazin-1-one113

A mixture of 113c (80 mg, 0.14 mmol), NaBH₄ (27 mg, 0.70 mmol), andmethanol (10 mL) was stirred at room temperature for 0.5 h. The mixturewas quenched with water (2 mL) and concentrated under reduced pressure.The residue was purified with reverse-phase prep-HPLC to afford 113 (57mg, 71%). MS-ESI: [M+H]⁺ 528.3. ¹H NMR (500 MHz, DMSO-d₆) δ 9.26 (s,1H), 8.76 (d, J=2.5 Hz, 1H), 8.67 (s, 1H), 8.59 (d, J=5.0 Hz, 1H), 8.54(d, J=2.5 Hz, 1H), 8.31 (d, J=6.0 Hz, 1H), 7.90 (d, J=1.5 Hz, 1H),7.79-7.76 (m, 1H), 7.69 (d, J=2.0 Hz 1H), 7.54 (d, J=5.0, Hz 1H),7.34-7.32 (m, 1H), 4.94 (t, J=5.0 Hz, 1H), 4.42-4.40 (m, 2H), 3.62 (s,3H), 1.39 (s, 9H).

Example 114a5-Bromo-1-methyl-3-(2-methylpyrimidin-4-ylamino)pyridin-2(1H)-one 114a

Following the procedure in Example 113a, and starting with2-methylpyrimidin-4-amine (2.0 g, 18.3 mmol) and3,5-dibromo-1-methylpyridin-2(1H)-one (9.6 g, 36 mmol) afforded 114a asa yellow solid (2.3 g, 43.4%). MS: [M+H]⁺ 295. ¹H NMR (500 MHz, DMSO-d₆)δ 9.20 (s, 1H), 8.78 (s, 1H), 8.26 (d, J=4.5 Hz, 1H), 7.68 (s, 1H), 7.18(d, J=4.5 Hz, 1H), 3.59 (s, 3H), 2.52 (s, 3H).

Example 114b1-Methyl-3-(2-methylpyrimidin-4-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one114b

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with bis(pinacolato) diboron(689 mg, 2.61 mmol), 1,4-dioxane (30 mL), 114a (307 mg, 1.04 mmol),Pd₂(dba)₃ (47 mg, 0.050 mmol), X-phos (48 mg, 0.10 mmol), and potassiumacetate (305 mg, 3.12 mmol). The mixture was heated at 65° C. for 6 h.It was then filtered and the filtrate was evaporated in vacuo to afford114b (300 mg, 84%) as a brown solid. MS: [M+H]⁺ 342.2

Example 114c2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(2-methylpyrimidin-4-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde114c

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 114b (236 mg, 0.69mmol),2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (250 mg, 0.69 mmol), PdCl₂(dppf) (29 mg, 0.035 mmol), K₃PO₄ (296mg, 1.39 mmol), sodium acetate (114 mg, 1.39 mmol), acetonitrile (15mL), and water (1 mL). The system was evacuated and refilled with N₂.The reaction mixture was heated at 100° C. for 2 h. It was then cooledto room temperature and filtered. The filtrate was concentrated underreduced pressure and the resulting residue was purified by silica-gelcolumn chromatography eluting with 30:1 dichloromethane/methanol toafford 114c (134 mg, 36%) as a yellow solid. MS-ESI: [M+H]⁺ 540.2.

Example 1146-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[(2-methylpyrimidin-4-yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one114

At 0° C., to a suspension of2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(2-methylpyrimidin-4-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde114c (130 mg, 0.24 mmol) in methanol (20 mL) was added sodiumborohydride (27 mg, 0.72 mmol). The reaction mixture was stirred for 20minutes and then quenched with water (10 mL). It was then concentratedunder reduced pressure and the residue was extracted withdichloromethane (3×20 mL). The combined organic layer was dried andconcentrated under reduced pressure. The residue was purified byreverse-phase prep-HPLC to afford 114 (20 mg, 15%) as a white solid.MS-ESI: [M+H]⁺ 542.3. ¹H NMR (500 MHz, CDCl₃) 8.97 (d, J=2.5 Hz, 1H),8.71 (d, J=5.0 Hz, 1H, 8.36 (d, J=2.0 Hz, 1H), 8.28 (d, J=5.5 Hz, 1H),8.09-8.06 (m, 1H), 7.86 (d, J=2.5 Hz, 1H), 7.60-7.56 (m, 3H), 6.61 (d,J=6.0 Hz, 1H), 4.54-4.43 (m, 2H), 4.16-4.13 (m, 1H), 3.75 (s, 3H), 2.62(s, 3H), 1.46 (s, 9H).

Example 115a 5-Methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-amine115a

A solution of 1-methyl-4-piperidone (11.3 g, 100 mmol) in 2-propanol (80mL) was heated to 50° C. To the solution were sequentially added asolution of cyanamide (4.2 g, 100 mmol) in 2-propanol (25 mL) and sulfurpowder (3.2 g, 100 mmol). After a catalytic amount of pyrrolidine (1.3mL) was added, the resultant mixture was stirred at 50° C. for 2 hours.The reaction mixture was allowed to cool to room temperature and stirredovernight. It was then cooled to or below 10° C. in an ice-water bathand stirred for 1 hour at the same temperature. The precipitatedcrystals were collected by filtration and washed with 2-propanol (20mL). The wet crystals were dried in vacuo to afford 115a (10 g, 59%).MS: [M+H]⁺ 170. ¹H NMR (500 MHz, DMSO-d₆) δ 6.70 (s, 2H), 3.31 (s, 2H),2.61 (t, J=5.5 Hz, 2H), 2.45 (m, 2H), 2.33 (s, 3H).

Example 115b5-Bromo-1-methyl-3-(5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-ylamino)pyridin-2(1H)-one115b

Following the procedures described for compound 113a and starting with115a (4.0 g, 23.5 mmol) and 3,5-dibromo-1-methylpyridin-2(1H)-one (3.0g, 17.8 mmol) afforded 115b as a yellow solid (2.8 g, 44%). MS: [M+H]⁺357.

Example 115c1-Methyl-3-(5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one115c

Compound 115b (997 mg, 2.8 mmol) was dissolved in dioxane (50 mL),followed by additions of bis(pinacolato)diboron (3.0 g, 12.0 mmol),Pd₂(dba)₃ (128 mg, 0.14 mmol), X-phos (134 mg, 0.28 mmol), and potassiumacetate (823 mg, 8.4 mmol). After three cycles of vacuum/argon flush,the mixture was heated at 65° C. for 2 h. The mixture was cooled to roomtemperature and filtered. The filtrate was concentrated under pressureand the residual was washed with petroleum ether (2×10 mL) to afford115c as a yellow solid (968 mg, 86%), which was used in next stepwithout further purification. MS-ESI: [M+H]⁺ 403.2

Example 115d2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde115d

A round-bottomed flask equipped with a reflux condenser was charged with2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (144 mg, 0.40 mmol), 115c (240 mg, 0.60 mmol), PdCl₂(dppf) (20 mg,0.020 mmol), K₃PO₄ (180 mg, 0.80 mmol), sodium acetate trihydrate (120mg, 0.80 mmol), and acetonitrile/water (15 mL/1 mL). After three cyclesof vacuum/argon flush, the mixture was heated at 100° C. for 2 h. It wasthen cooled to room temperature and filtered. The filtrate wasevaporated under reduced pressure. The residue was purified withsilica-gel column chromatography eluting with 25:1dichloromethane/methanol to afford 115d as a yellow solid (100 mg, 42%).MS-ESI: [M+H]⁺ 600.3

Example 1156-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[(5-methyl-6,7-dihydro-4H-thiazolo[5,4-c]pyridin-2-yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one115

To a mixture of 115d (100 mg, 0.15 mmol) in methanol (6 mL) was addedNaBH₄ (18 mg, 0.45 mmol). The mixture was stirred at 30° C. for 1 h andthen quenched with water (10 mL). It was extracted with dichloromethane(3×30 mL) and the combined organic layer was concentrated under reducedpressure. The residue was purified by reverse-phase prep-HPLC to afford115 (35 mg, 36%) as a white solid. MS-ESI: [M+H]⁺ 602.2. ¹H NMR (500MHz, CDCl₃) δ 8.68 (d, J=5.0 Hz, 1H), 8.42 (d, J=2.5 Hz, 1H), 8.35 (d,J=2.5 Hz, 1H), 8.30 (s, 1H), 7.77 (d, J=2.0 Hz, 1H), 7.58-7.53 (m, 3H),4.49-4.45 (m, 2H), 4.10 (bs, 1H), 3.73 (s, 3H), 3.58 (s, 2H), 2.83-2.79(m, 4H), 2.52 (s, 3H), 1.45 (s, 9H).

Example 116a6-tert-Butyl-2-(4-chloro-3-(hydroxymethyl)pyridin-2-yl)-8-fluorophthalazin-1(2H)-one116a

To a solution of2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (2.0 g, 5.5 mmol) in methanol (30 mL) was added NaBH₄ (700 mg, 16.5mmol) at room temperature. The reaction mixture was stirred for 1 h andquenched with water (30 mL). It was then concentrated under reducedpressure and the residue was extracted with dichloromethane (3×30 mL).The combined organic phase was dried over anhydrous Na₂SO₄, filtered,and evaporated under reduced pressure to afford 116a as a white solid(1.8 g, 90%). MS: [M+H]⁺ 362.3.

Example 116b(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloropyridin-3-yl)methylAcetate 116b

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with 116a (1.6 g, 4.4 mmol), acetic anhydride (10mL), and triethylamine (1 mL). The reaction mixture was stirred at roomtemperature for 1 h and concentrated under reduced pressure. The residuewas purified by silica-gel column chromatography eluting with 30:1dichloromethane/methanol to afford 116b as a brown solid (1.4 g, 82%).MS: [M+H]⁺ 404.3.

Example 116c3-(Acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicAcid 116c

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 116b (1.0 g, 2.5 mmol),Pin₂B₂ (3.2 g, 12.5 mmol), Pd(dppf)Cl₂ (75 mg, 0.125 mmol), X-phos (75mg, 0.25 mmol), potassium acetate (750 mg, 7.5 mmol), and dioxane (60mL). After three cycles of vacuum/argon flush, the mixture was heated at65° C. for 15 h. It was then cooled to room temperature and filtered.The filtrate was concentrated under reduced pressure and the resultingresidue was washed with 3:1 petroleum ether/ethyl acetate (10 mL) toafford 116c as yellow solid (1.0 g, LCMS purity: 75%). MS: [M+H]⁺ 414.2.

Example 116d6-Chloro-2-methyl-4-({5-[(morpholin-4-yl)carbonyl]pyridin-2-yl}amino)-2,3-dihydropyridazin-3-one116d

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (40 mL),(6-aminopyridin-3-yl)(morpholino)methanone (2.07 g, 10.0 mmol),4-bromo-6-chloro-2-methylpyridazin-3(2H)-one (3.35 g, 15.0 mmol),Pd₂(dba)₃ (915 mg, 1.0 mmol), XantPhos (578 mg, 1.0 mmol), and cesiumcarbonate (6.52 g, 20 mmol). After three cycles of vacuum/argon flush,the mixture was heated at 100° C. for 8 h. It was then cooled to roomtemperature and filtered. The solid was washed with dichloromethane(2×20 mL). The combined filtrate was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to afford 116d (2.45 g, 51%) as ayellow solid. MS: [M+H]⁺ 350.1

Example 116e(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-(morpholine-4-carbonyl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridazin-3-yl)pyridin-3-yl)methylAcetate 116e

A 50-mL round-bottomed flask equipped with a reflux condenser wascharged with 116d (175 mg, 0.50 mmol),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicacid 116c (300 mg, 0.75 mmol), Pd(dppf)Cl₂ (25 mg, 0.025 mmol), K₃PO₄(220 mg, 1.0 mmol), sodium acetate trihydrate (150 mg, 1.0 mmol), andacetonitrile/water (20/1 mL). After three cycles of vacuum/argon flush,the mixture was heated at 100° C. for 2 h. It was then filtered and thefiltrate was evaporated under reduced pressure. The residue was purifiedby silica-gel column chromatography eluting with 25:1dichloromethane/methanol to afford 116e as a yellow solid (150 mg, 45%).MS-ESI: [M+H]⁺ 683.3

Example 1166-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[[5-(morpholine-4-carbonyl)-2-pyridyl]amino]-6-oxo-pyridazin-3-yl]-2-pyridyl]phthalazin-1-one116

A mixture of 116e (150 mg, 0.20 mmol) and lithium hydroxide (85 mg, 2.0mmol) in THF/i-propanol (5/3 mL) and water (2 mL) was stirred at 30° C.for 1 h. The mixture was evaporated under reduce pressure and theresidue was extracted with ethyl acetate (3×20 mL). The combined ethylacetate extract was concentrated under reduced pressure and the residuewas purified by reverse-phase prep-HPLC to afford 116 (80 mg, 80%) as awhite solid. MS-ESI: [M+H]⁺ 641.3. ¹H NMR (500 MHz, CDCl₃) δ 8.78 (s,1H), 8.75 (d, J=5.0 Hz, 1H), 8.47 (s, 1H), 8.45 (d, J=2.0 Hz, 1H), 8.34(d, J=3.0 Hz, 1H), 7.79 (dd, J=2.0, 8.0 Hz, 1H), 7.64 (d, J=4.5 Hz, 1H),7.57 (s, 1H), 7.52 (dd, J=1.5, 12.5 Hz, 1H), 7.02 (d, J=8.5 Hz, 1H),4.57-4.55 (m, 2H), 3.94 (s, 3H), 3.81-3.75 (m, 6H), 1.64-1.62 (m, 2H),1.44 (s, 9H).

Example 117a (S)-tert-Butyl4-(6-(6-Chloro-2-methyl-3-oxo-2,3-dihydropyridazin-4-ylamino)pyridin-3-yl)-3-methylpiperazine-1-carboxylate117a

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with (S)-tert-butyl4-(6-aminopyridin-3-yl)-3-methylpiperazine-1-carboxylate 105b (2.5 g,8.5 mmol), 4-bromo-6-chloro-2-methylpyridazin-3(2H)-one (2.2 g, 10.0mmol), XantPhos (240 mg, 0.40 mmol),tris(dibenzylideneacetone)dipalladium(0) (360 mg, 0.40 mmol), Cs₂CO₃(5.5 g, 17 mmol), and 1,4-dioxane (100 mL). After three cycles ofvacuum/argon flush, the mixture was heated at 100° C. for 2.5 h. It wasthen cooled to room temperature and filtered. The filtrate wasconcentrated under reduced pressure and the resulting residue waspurified by silica-gel column chromatography eluting withdichloromethane/methanol (40:1 to 30:1) to afford 117a as a pale yellowsolid (3.2 g, 86%). MS-ESI: [M+H]⁺ 435.1.

Example 117b(S)-6-Chloro-2-methyl-4-(5-(2-methylpiperazin-1-yl)pyridin-2-ylamino)pyridazin-3(2H)-one117b

A mixture of 117a (3.0 g, 6.9 mmol) and 4.0M HCl/ethanol (20 mL) wasstirred at room temperature for 2 h. The mixture was then concentratedunder reduced pressure to afford crude 117b as a yellow solid (2.5 g,98%), which was used in the next step without further purification.MS-ESI: [M+H]⁺ 335.1.

Example 117c(S)-6-Chloro-2-methyl-4-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)pyridazin-3(2H)-one117c

A mixture of 117b (2.3 g, 6.8 mmol), oxetan-3-one (1.4 g, 20.0 mmol),NaBH₃CN (620 mg, 10 mmol), and zinc chloride (1.36 g, 10 mmol) inmethanol (20 mL) was stirred at 50° C. for 3 hours. The mixture wasadded to water (40 mL) and concentrated under reduced pressure. Theresidue was extracted with dichloromethane three times. The combinedorganic layer was dried and concentrated under reduced pressure. Theresidue was purified by silica-gel column chromatography eluting with50:1 dichloromethane/methanol to afford 117c (2.0 g, 75%). MS-ESI:[M+H]⁺ 391.2.

Example 117d(S)-(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridazin-3-yl)pyridin-3-yl)methylAcetate 117d

A round-bottomed flask equipped with a reflux condenser was charged with117c (200 mg, 0.50 mmol),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-yl-boronicacid 116c (300 mg, 0.75 mmol), Pd(dppf)Cl₂ (25 mg, 0.025 mmol), K₃PO₄(220 mg, 1.0 mmol), sodium acetate trihydrate (150 mg, 1.0 mmol), andacetonitrile/water (20/1 mL). After three cycles of vacuum/argon flush,the mixture was heated at 100° C. for 2 h. It was then cooled to roomtemperature and filtered. The filtrate was evaporated under reducedpressure and the residue was purified with silica-gel columnchromatography eluting with 25:1 dichloromethane/methanol to afford 117das a yellow solid (110 mg, 42%). MS-ESI: [M+H]⁺ 724.4

Example 1176-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-6-oxo-pyridazin-3-yl]-2-pyridyl]phthalazin-1-one117

A mixture of 117d (110 mg, 0.15 mmol) and lithium hydroxide (65 mg, 1.5mmol) in THF/i-propanol (5/3 mL) and water (2 mL) was stirred at 30° C.for 1 h. The mixture was evaporated under reduced pressure and theresidue was extracted with ethyl acetate (2×20 mL). The combined ethylacetate extract was concentrated under reduced pressure and the residuewas purified by reverse-phase prep-HPLC to afford 117 (100 mg, 95%) as ayellow solid. MS-ESI: [M+H]⁺ 682.4. ¹H NMR (500 MHz, CDCl₃) δ 8.73 (d,J=5.0 Hz, 1H), 8.60 (s, 1H), 8.33 (d, J=2.5 Hz, 1H), 8.24 (s, 1H), 8.07(s, 1H), 7.65 (d, J=5.0 Hz, 1H), 7.56-7.51 (m, 2H), 7.34 (s, 1H), 6.96(d, J=9.0 Hz, 1H), 4.73-4.55 (m, 5H), 3.92 (m, 4H), 3.73-3.74 (m, 1H),3.55-3.53 (m, 1H), 3.16-3.15 (m, 2H), 2.64-2.39 (m, 4H), 1.44 (s, 9H),1.09-1.07 (m, 3H).

Example 118a5-Bromo-1-methyl-3-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2(1H)-one 118a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and reflux condenser was charged with 1,4-dioxane (15 mL),5-methyl-1H-pyrazol-3-amine (1 g, 10 mmol) (1),3,5-dibromo-1-methylpyridin-2(1H)-one (4 g, 15 mmol) (2), and cesiumcarbonate (6.4 g, 20 mmol). Xantphos (400 mg, 0.8 mmol) and Pd₂(dba)₃(700 mg, 0.8 mmol) were added, and the reaction mixture was heated at100° C. for 5 h. After this time the reaction was cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure and the residue was purified on flush column eluting withdichloromethane:methanol (20:1) to afford 118a (1.0 g, 35%). MS: [M+H]⁺283.

Example 118b5-Bromo-3-(1-ethyl-5-methyl-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one118b

A 100-mL round-bottomed flask was charged with 118a (800 mg, 2.83 mmol),bromoethane (216 mg, 1.98 mmol), K₂CO₃ (780 mg, 5.66 mmol), and DMF (20mL). The mixture was heated at 85° C. overnight. It was then filteredand the filtrate was evaporated in vacuo. The residue was purified bysilica-gel column chromatography eluting with 1:20methanol/dichloromethane to afford 118b as a red solid (298 mg, 37%).MS-ESI: [M+H]⁺ 311.0. ¹H NMR (500 MHz, DMSO-d₆) δ 8.28 (s, 1H), 7.99 (d,J=2.5 Hz, 1H), 7.35 (d, J=2.5 Hz, 1H), 5.85 (s, 1H), 3.98-3.94 (m, 2H),3.48 (s, 3H), 2.19 (s, 3H), 1.27 (t, J=7.0 Hz, 3H).

Example 118c(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(1-ethyl-5-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 118c

A 50-mL round-bottomed flask equipped with a reflux condenser wascharged with 118b (103 mg, 0.33 mmol),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicacid 116c (136 mg, 0.33 mmol), PdCl₂(dppf) (27 mg, 0.033 mmol), K₃PO₄(171 mg, 0.66 mmol), sodium acetate (54 mg, 0.66 mmol), acetonitrile (10mL), and water (0.5 mL). After three cycles of vacuum/argon flush, themixture was heated at 100° C. for 3 h. It was then filtered and thefiltrate was in vacuo. The residue was purified by silica-gel columnchromatography eluting with 1:20 methanol/dichloromethane to afford 118cas a yellow solid (80 mg, 41%). MS-ESI: [M+H]⁺ 600.2

Example 1186-tert-butyl-2-[4-[5-[(1-ethyl-5-methyl-pyrazol-3-yl)amino]-1-methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one118

A mixture of 118c (80 mg, 0.13 mmol), lithium hydroxide (13 mg, 0.53mmol), THF (6 mL), i-propanol (4 mL) and water (2 mL) was stirred atroom temperature for 0.5 h. The mixture was concentrated under reducedpressure and diluted with water (5 mL). It was then extracted withdichloromethane (2×10 mL). The combined dichloromethane extract wasconcentrated under reduced pressure and the residue was purified withreverse-phase prep-HPLC to afford 118 (20 mg, 26%) as a white solid.MS-ESI: [M+H]⁺ 558.3. ¹H NMR (500 MHz, DMSO-d₆) δ 8.56 (d, J=5.0 Hz,1H), 8.53 (d, J=2.5 Hz, 1H), 8.10 (s, 1H), 8.05 (d, J=2.5 Hz, 1H), 7.90(d, J=2.0 Hz, 1H), 7.79-7.76 (m, 1H), 7.50 (d, J=5.0 Hz, 1H), 7.40 (d,J=2.5 Hz, 1H), 5.88 (s, 1H), 4.92 (t, J=5.0 Hz 1H), 4.46-4.45 (m, 2H),3.91 (q, J=7.5 Hz, 2H), 3.59 (s, 3H), 2.19 (s, 3H), 1.40 (s, 9H), 1.27(t, J=7.0 Hz, 3H).

Example 119a5-Bromo-3-(1,5-dimethyl-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one119a

A solution of5-bromo-1-methyl-3-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2(1H)-one 118a(2.8 g, 9.9 mmol) in anhydrous DMF (10 mL) was treated with 60%dispersion of NaH in mineral oil (0.51 g, 13 mmol) while stirring undernitrogen. After the resulting effervescence ceased, the reaction wasstirred for an additional 30 minutes. At this time the reaction wastreated with iodomethane (0.98 g, 7.0 mmol) with continued stirringunder nitrogen for 2 hours. Water (50 mL) was added slowly and themixture was filtered. The filtrate was extracted with ethyl acetate(3×30 mL). The combined extract was concentrated under reduced pressureand the residue was purified by flush column chromatography eluting with3:1 petroleum ether/ethyl acetate to afford 119a (0.70 g, 24%). MS:[M+H]⁺ 297.

Example 119b(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(1,5-dimethyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 119b

A 50-mL round-bottomed flask equipped with a reflux condenser wascharged with 119a (180 mg, 0.50 mmol),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridine-4-ylboronicacid 116c (207 mg, 0.50 mmol), Pd(dppf)Cl₂ (41 mg, 0.050 mmol), K₃PO₄(212 mg, 1.0 mmol), sodium acetate (82 mg, 1.0 mmol), water (0.5 mL),and acetonitrile (10 mL). After three cycles of vacuum/argon flush, themixture was heated at reflux for 2 h. It was then cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure and the resulting residue was purified by silica-gel columnchromatography eluting with 30:1 dichloromethane/methanol to afford 119bas a white solid (175 mg, 60%). MS-ESI: [M+H]⁺ 586.4

Example 1196-tert-butyl-2-[4-[5-[(1,5-dimethylpyrazol-3-yl)amino]-1-methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one119

A mixture of 119b (150 mg, 0.26 mmol) and lithium hydroxide (61.4 mg,2.6 mmol) in i-propanol/THF (1:1, 4 mL) and water (1 mL) was stirred at30° C. for 1 h. The mixture was evaporated in vacuo and the residue wasdiluted with water (5 mL). It was then extracted with ethyl acetate(2×10 mL). The combined ethyl acetate extract was concentrated underreduced pressure and the residue was purified by reverse-phase prep-HPLCto afford 119 (75.0 mg, 54%) as a white solid. MS-ESI: [M+H]⁺ 544.3. ¹HNMR (500 MHz, CDCl₃) δ 8.66 (d, J=5.0 Hz, 1H), 8.35 (d, J=2.0 Hz, 1H),7.97 (d, J=2.0 Hz, 1H), 7.59-7.53 (m, 4H), 7.37 (s, 1H), 5.74 (s, 1H),4.51 (s, 2H), 4.07 (s, 1H), 3.72 (s, 3H), 3.70 (s, 3H), 2.25 (s, 3H),1.46 (s, 9H).

Example 120a5-Bromo-1-methyl-3-(5-methylthiazol-2-ylamino)pyridin-2(1H)-one 120a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (50 mL),5-methylthiazol-2-amine (2.28 g, 20.0 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (5.34 g, 20.0 mmol) cesiumcarbonate (13.0 g, 40.0 mmol), xantphos (1.16 g, 2.0 mmol), andtris(dibenzylideneacetone)dipalladium(0) (916 mg, 1.0 mmol). The systemwas subjected to three cycles of vacuum/argon flush and heated at refluxfor 5 h. It was then cooled to room temperature and filtered. The solidwas washed with dichloromethane (3×50 mL). The combined filtrate wasconcentrated under reduced pressure and the residue was washed withacetonitrile (30 mL) to afford 120a (5 g, crude, 83%) as a yellow solid.MS-ESI: [M+H]⁺ 300.1.

Example 120b1-Methyl-3-(5-methylthiazol-2-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one120b

A 250-mL round-bottomed flask equipped with a magnetic stirrer and areflux condenser was charged with 120a (5.0 g, 16.6 mmol), Pin₂B₂ (21.1g, 83.0 mmol), tris(dibenzylideneacetone)dipalladium(0) (760 mg, 0.83mmol), x-phos (810 mg, 1.7 mmol), potassium acetate (4.9 g, 50 mmol),and 1,4-dioxane (80 mL). The reaction mixture was subjected to threecycles of vacuum/argon flush and heated at 65° C. for 3 h. It was thencooled to room temperature and filtered. The filtrate was concentratedunder reduced pressure and the resulting residue was washed withpetroleum ether to afford 120b (20.0 g, crude) as brown solid, which wasused in next step without further purification. MS-ESI: [M+H]⁺ 348.2

Example 120c2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-methylthiazol-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde120c

A 25-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 120b (174 mg, 0.50mmol),2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (180 mg, 0.50 mmol), K₃PO₄ (212 mg, 1.0 mmol), sodium acetate (82mg, 1.0 mmol), 1,1′-bis(diphenylphosphino)ferrocenedichloropalladium(II)(18 mg, 0.025 mmol), acetonitrile (8 mL), and water (0.5 mL). Thereaction mixture was subjected to three cycles of vacuum/argon flush andheated at 100° C. for 1 h. Analysis of the reaction mixture by LCMSshowed complete conversion to the desired product. The reaction mixturewas cooled to room temperature and filtered. The filtrate wasconcentrated under reduced pressure. The residue was diluted withdichloromethane (50 mL) and water (30 mL). The water layer was extractedwith dichloromethane (2×30 mL). The combined organic extract was driedover Na₂SO₄, filtered, and concentrated under reduced pressure. The darkresidue was purified by silica-gel column chromatography eluting withdichloromethane/methanol (80:1 to 30:1) to afford 120c (150 mg, 55%) asa yellow solid. MS-ESI: [M+H]⁺ 545.3

Example 1206-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[(5-methylthiazol-2-yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one120

To a solution of 120c (98 mg, 0.18 mmol) in methanol/dichloromethane(3/3mL) was added NaBH₄ (21 mg, 0.55 mmol) at room temperature. After thereaction was stirred for 1 h, LCMS indicated the reaction was complete.The mixture was quenched with water (5 mL) and concentrated underreduced pressure. The residue was extracted with dichloromethane (3×15mL). The combined organic layer was washed with brine (20 mL), driedover Na₂SO₄, filtered, and concentrated under reduced pressure. Theresidue was purified by reverse-phase prep-HPLC to afford 120 (30 mg,31%) as a white solid. MS-ESI: [M+H]⁺ 546.7. ¹H NMR (500 MHz, DMSO-d₆) δ9.92 (s, 1H), 8.58 (d, J=5.5 Hz, 1H), 8.56 (d, J=2.5 Hz, 1H), 8.54 (d,J=2.0 Hz, 1H), 7.91 (d, J=2.0 Hz, 1H), 7.78 (d, J=13.0 Hz, 1H), 7.56 (d,J=2.0 Hz, 1H), 7.51 (d, J=5.0 Hz, 1H), 6.96 (d, J=1.0 Hz, 1H), 4.91 (bs,1H), 4.42-4.41 (m, 2H), 3.60 (s, 3H), 2.28 (d, J=1.0 Hz, 3H), 1.40 (s,9H).

Example 121a(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-methyl-1H-pyrazol-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 121a

A 50-mL round-bottomed flask equipped with a reflux condenser wascharged with5-bromo-1-methyl-3-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2(1H)-one 118a(142 mg, 0.50 mmol),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicacid 116c (310 mg, 0.75 mmol), Pd(dppf)Cl₂ (18 mg, 0.025 mmol), K₃PO₄(212 mg, 1.0 mmol), sodium acetate (82 mg, 1.0 mmol), acetonitrile (10mL), and water (0.2 mL). After three cycles of vacuum/argon flush, themixture was heated at 100° C. for 2 h. It was then filtered and thefiltrate was evaporated under reduced pressure. The residue was purifiedby silica-gel column chromatography eluting with 25:1dichloromethane/methanol to afford 121a as a brown solid (100 mg, 35%).MS-ESI: [M+H]⁺ 572.3

Example 1216-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[(5-methyl-1H-pyrazol-3-yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one121

A mixture of 121a (86 mg, 0.15 mmol) and lithium hydroxide (36 mg, 1.5mmol) in THF/i-propanol (5:3, 8 mL) and water (2 mL) was stirred at 30°C. for 1 h. The mixture was evaporated under reduced pressure and theresidue was diluted with water (5 mL). It was then extracted with ethylacetate (2×10 mL). The combined ethyl acetate extract was concentratedunder reduced pressure and the residue was purified by reverse-phaseprep-HPLC to afford 121 (24 mg, 30%) as a white solid. MS-ESI: [M+H]⁺530.3. ¹H NMR (500 MHz, CDCl₃) δ 8.65 (d, J=5.0 Hz, 1H), 8.35 (d, J=2.0Hz, 1H), 8.04 (d, J=2.5 Hz, 1H), 7.58-7.53 (m, 4H), 7.44 (s, 1H), 5.78(s, 1H), 4.51-4.49 (m, 2H), 3.73 (s, 3H), 2.03 (s, 3H), 1.45 (s, 9H).

Example 122a5-Bromo-3-(5-ethyl-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one 122a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (80 mL),5-ethyl-1H-pyrazol-3-amine (3.33 g, 30.0 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (9.6 g, 36 mmol), and cesiumcarbonate (19.5 g, 60 mmol). After bubbling nitrogen through thesuspension for 10 minutes, Xantphos (1.73 mg, 3.0 mmol) andtris(dibenzylideneacetone)dipalladium(0) (1.36 mg, 1.5 mmol) were added.The system was subjected to three cycles of vacuum/argon flush andheated at reflux for 2 h. It was then filtered immediately. The solidwas washed with dioxane (3×30 mL) and the combined filtrate wasconcentrated under reduced pressure. The residue was purified bysilica-gel column chromatography eluting with petroleum ether/ethylacetate (2:1 to 1:2) to afford 122a (3.8 g, 43%) as a red solid. MS-ESI:[M+H]⁺ 297.0

Example 122b5-Bromo-3-(5-ethyl-1-methyl-1H-pyrazol-3-ylamino)-1-methylpyridin-2(1H)-one122b

To a mixture of 122a (598 mg, 2.0 mmol) in DMF (10 mL) was added NaH (64mg, 1.6 mmol) at 0° C. and the mixture was stirred for 0.5 h. To themixture was added iodomethane (227 mg, 1.6 mmol) in DMF dropwise at 0°C. The mixture was stirred for 0.5 h and quenched with water (20 mL). Itwas then extracted with dichloromethane (3×30 mL). The combined extractwas concentrated under reduced pressure and the residue was purifiedwith silica-gel column chromatography eluting with petroleum ether/ethylacetate (5:1 to 2:1) to afford 122b (360 mg, 58%) as a light yellowsolid. MS-ESI: [M+H]⁺ 311.1

Example 122c(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(5-ethyl-1-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 122c

A 50-mL round-bottomed flask equipped with a reflux condenser wascharged with 122b (310 mg, 1.0 mmol),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicacid 116c (1.24 g, 3.0 mmol), K₃PO₄ (424 mg, 2.0 mmol), sodium acetate(164 mg, 2.0 mmol),1,1′-bis(diphenylphosphino)ferrocenedichloropalladium(II) (73 mg, 0.10mmol), acetonitrile (10 mL), and water (0.5 mL). The system was subjectto three cycles of vacuum/nitrogen flush and heated at 100° C. under N₂protection for 2.5 h. The reaction mixture was cooled to roomtemperature and concentrated under reduced pressure. The residue wasdiluted with dichloromethane (50 mL) and water (50 mL). The aqueouslayer was separated and extracted with dichloromethane (3×20 mL). Thecombined organic layer was dried over Na₂SO₄, filtered, and concentratedunder reduced pressure. The dark residue was purified with silica-gelcolumn chromatography eluting with 60:1 dichloromethane/methanol toafford 122c (150 mg, 25%) as a yellow oil. MS-ESI: [M+H]⁺ 600.0

Example 1226-tert-butyl-2-[4-[5-[(5-ethyl-1-methyl-pyrazol-3-yl)amino]-1-methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one122

To a solution of 122c (150 mg, 0.25 mmol) in THF/i-propanol/water(2.5/1/0.5 mL) was added lithium hydroxide (70 mg, 2.5 mmol) at 35° C.After the reaction was stirred for 3 h, LCMS indicated the reaction wascompleted. The mixture was poured into water (15 mL) and extracted withdichloromethane (3×20 mL). The combined organic layer was washed withbrine (30 mL), dried over Na₂SO₄, filtered, and concentrated underreduced pressure. The residue solid was purified by reverse-phaseprep-HPLC to afford 122 (49 mg, 35%) as a white solid. MS-ESI: [M+H]⁺557.8. ¹H NMR (500 MHz, CDCl₃) δ 8.59 (d, J=5.0 Hz, 1H), 8.51 (d, J=2.5Hz, 1H), 7.92 (d, J=2.0 Hz, 1H), 7.88 (d, J=1.5 Hz, 1H), 7.73 (m, 1H),7.64 (d, J=5.0 Hz, 1H), 7.41 (d, J=1.5 Hz, 1H), 5.92 (s, 1H), 4.59 (s,2H), 3.72 (s, 3H), 3.68 (s, 3H), 2.64 (m, 2H), 1.47 (s, 9H), 1.27 (t,J=2.5, 3H)

Example 123a 1-Ethyl-4-nitro-1H-pyrazole 123a

To a solution of 4-nitro-1H-pyrazole (5.0 g, 44.2 mmol) in anhydrous DMF(100 mL) was NaH (60% in oil) (1.94 g, 48.6 mmol) while being stirred at−25° C. under nitrogen. The mixture was stirred for 30 min beforebromoethane (5.30 g, 48.6 mmol) was added. The mixture was continued tostir under nitrogen at −25° C. for 6 h. The solution was diluted withethyl acetate (100 mL), washed with water (2×50 mL) and brine solution(50 mL), dried over magnesium sulfate, and filtered. The filtrate wasconcentrated under reduced pressure to afford crude 123a (5.0 g, 80%),which was used in next step without further purification. MS-ESI: [M+H]⁺142.0

Example 123b 1-Ethyl-1H-pyrazol-4-amine 123b

A 100-mL single-neck round-bottomed flask was purged with hydrogen andcharged with 123a (4.5 g, 31.9 mmol), 10% palladium on carbon (10% wet,2.0 g), and methanol (50 mL). The mixture was stirred at roomtemperature for 6 h. The catalyst was removed by filtration through apad of CELITE® and the filtrate was concentrated under reduced pressureto afford 123b (3.3 g, 93%). MS-ESI: [M+H]⁺ 112.0

Example 123c5-Bromo-3-(1-ethyl-1H-pyrazol-4-ylamino)-1-methylpyrazin-2(1H)-one 123c

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and reflux condenser was charged with 123b (500 mg, 4.5 mmol),3,5-dibromo-1-methylpyrazin-2(1H)-one (2.40 g, 9.0 mmol), DIPethylacetate (3 mL), and isopropanol (50 mL). The mixture was heated at 100°C. for 2 h. It was then cooled to room temperature and filtered. Thefiltrate was concentrated under reduced pressure to afford 123c (802 mg,60%) as a white solid. MS-ESI: [M+H]⁺ 298.0

Example 123d(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(6-(1-ethyl-1H-pyrazol-4-ylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)pyridin-3-yl)methylAcetate 123d

A 50-mL round-bottomed flask equipped with a magnetic stirrer and areflux condenser was charged with 123c (151 mg, 0.51 mmol),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicacid 116c (206 mg, 0.50 mmol), PdCl₂(dppf) (22 mg, 0.030 mmol), K₃PO₄(216 mg, 1.02 mmol), sodium acetate (84 mg, 1.02 mmol), acetonitrile (10mL), and water (0.5 mL). After three cycles of vacuum/argon flush, themixture was heated at 100° C. for 3 h. It was then filtered and thefiltrate was evaporated under reduced pressure. The residue was purifiedby silica-gel column chromatography eluting with 1:3 petroleum/ethylacetate to afford 123d as a yellow solid (158 mg, 53%). MS-ESI: [M+H]⁺587.2

Example 1236-tert-butyl-2-[4-[6-[(1-ethylpyrazol-4-yl)amino]-4-methyl-5-oxo-pyrazin-2-yl]-3-(hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one123

A mixture of 123d (152 mg, 0.26 mmol) and lithium hydroxide (61 mg, 2.56mmol) in i-propanol/THF (1:1, 10 mL) and water (3 mL) was stirred atroom temperature for 1 h. The mixture was evaporated under reducedpressure and the residue was extracted with ethyl acetate (2×10 mL). Thecombined ethyl acetate extract was concentrated under reduced pressureand the residue was purified by reverse-phase prep-HPLC to afford 123(55 mg, 39%) as a yellow solid. MS-ESI: [M+H]⁺ 545.2. ¹H NMR (500 MHz,DMSO-d₆) δ 9.72 (s, 1H), 8.58 (d, J=5.0 Hz, 1H), 8.54 (d, J=2.5 Hz, 1H),8.18 (s, 1H), 7.90 (d, J=2.0 Hz, 1H), 7.80-7.75 (m, 1H), 7.74 (s, 1H),7.71 (d, J=5.0 Hz, 1H), 7.51 (s, 1H), 4.90 (t, J=5.0 Hz, 1H), 4.61-4.53(m, 2H), 4.09 (q, J=7.5 Hz, 2H), 3.54 (s, 3H), 1.41 (s, 9H), 1.35 (t,J=6.0 Hz, 3H).

Example 124a (3-Nitro-1H-pyrazol-5-yl)methanol 124a

A 3-L three-neck round-bottomed flask equipped with a mechanicalstirrer, addition funnel and nitrogen inlet was purged with nitrogen andcharged with 3-nitropyrazole-5-carboxylic acid (28.0 g, 178 mmol) andTHF (420 mL) and cooled to −5° C. using an ice/acetone bath. Borane-THFcomplex solution (1.0 M, 535 mL, 535 mmol) was added at a rate thatmaintained the internal reaction temperature below 5° C. After theaddition was complete the cooling bath was removed and the reaction wasstirred at room temperature for 18 h. After this time the reaction wascooled to −5° C. using an ice/acetone bath, water (70 mL) and 4Nhydrochloric acid (70 mL) was added and the reaction was stirred atreflux for 1 h in order to destroy the borane complex with pyrazole. Thereaction was cooled to room temperature and concentrated under reducedpressure to a volume of approximately 30 mL. Ethyl acetate (175 mL) wasadded and the mixture stirred for 15 min. The aqueous layer wasseparated and extracted with ethyl acetate (4×200 mL). The combinedorganic layers were washed with saturated aqueous sodium bicarbonate(2×50 mL), brine (50 mL) and dried over sodium sulfate, the drying agentwas removed by filtration, and the filtrate concentrated under reducedpressure to afford 124a in a 94% yield (24.0 g) as a light yellow solid:¹H NMR (300 MHz, DMSO-d₆) δ 13.90 (br s, 1H), 6.87 (s, 1H), 5.58 (t, 1H,J=5.4 Hz), 4.53 (d, 2H, J=5.1 Hz); MS (ESI+) m/z 144.0 (M+H)

Example 124b (1-(2-Bromoethyl)-3-nitro-1H-pyrazol-5-yl)methanol 124b

A 1-L three-necked round-bottomed flask equipped with a mechanicalstirrer and thermoregulator was purged with nitrogen and charged with124a (25.0 g, 175 mmol), DMF (250 mL), and cesium carbonate (70.0 g, 215mmol) was heated at 104° C. for 5 min. The reaction mixture was thencooled to 0° C. using an ice/acetone bath and dibromoethane (329 g, 1.75mol) was added portionwise (no exotherm). The reaction was stirred at 0°C. for 1 then at room temperature for 4 h. After this time a solution ofKH₂PO4 (40 g) in water (400 mL) was added slowly. The reaction mixturewas stirred at room temperature for 30 min. Ethyl acetate (450 mL) wasadded and the aqueous layer was separated and extracted with ethylacetate (2×100 mL). The combined organic layers were washed with water(200 mL), brine (200 mL), dried over sodium sulfate, and the dryingagent was removed by filtration. The filtrate was concentrated underreduced pressure to afford an 86% yield (37.5 g) of crude 124b as anorange oil: ¹H NMR (300 MHz, CDCl₃) δ 6.85 (s, 1H), 4.82 (d, 2H, J=5.4Hz), 4.66 (t, 2H, J=6.3 Hz), 3.83 (t, 2H, J=6.3 Hz); MS (ESI+) m/z 249.9(M+H).

Example 124c 1-(2-Bromoethyl)-5-(bromomethyl)-3-nitro-1H-pyrazole 124c

A 500-mL three-necked round-bottomed flask equipped with a magneticstirrer, nitrogen inlet and reflux condenser was purged with nitrogenand charged with 124b (37.0 g, 148 mmol) and chloroform (160 mL). Thereaction was cooled to −5° C. using an ice/acetone bath and phosphoroustribromide (40.0 g, 148 mmol) was added portionwise. The cooling bathwas removed and the reaction stirred at reflux for 2 h. After this time,the reaction was cooled to −5° C. and saturated aqueous sodiumbicarbonate (250 mL) was added until a pH of 8.5 was reached. Themixture was extracted with ethyl acetate (3×150 mL) and the combinedorganic layers were washed with saturated aqueous sodium carbonate (2×50mL), brine (75 mL), dried over sodium sulfate and the drying agent wasremoved by filtration. The filtrate was concentrated under reducedpressure to afford a yellow residue that was dissolved with gentleheating in methylene chloride (60 mL). Hexanes (approximately 20 mL) wasadded and the solution became cloudy. The mixture was heated until asolid precipitate formed, methylene chloride (9 mL) was added and thesolution became clear. The solution was left to cool to room temperatureand after 4 h the resulting crystals were collected by vacuumfiltration. The filter cake was washed with a ice cold 1:2 mixture ofmethylene chloride:hexanes (2×20 mL) to afford1-(2-bromoethyl)-5-(bromomethyl)-3-nitro-1H-pyrazole (19.7 g). Thecombined filtrates were evaporated and the procedure was performed againto afford an additional 9.70 g of1-(2-bromoethyl)-5-(bromo-methyl)-3-nitro-1H-pyrazole. The solids werecombined and dried under high vacuum for 18 h to afford a 57% yield(26.0 g) of 124c as white crystals: mp 95-97° C.; ¹H NMR (300 MHz,CDCl₃) δ 6.93 (s, 1H), 4.63 (t, 2H, J=6.0 Hz), 4.54 (s, 2H), 3.86 (t,2H, J=6.0 Hz).

Example 124d 2-Nitro-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine 124d

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with 124c (3.0 g, 9.64 mmol) in THF (35 mL) andaqueous ammonia (135 mL, 25-28%). The mixture was stirred at roomtemperature for 72 h under nitrogen. The reaction mixture was thenconcentrated under reduced pressure and the resulting residue waspartitioned between ethyl acetate (100 mL) and water (100 mL). Theaqueous layer was extracted with ethyl acetate (2×50 mL). The combinedorganic layer was washed with 10% potassium carbonate (2×100 mL), brine(200 mL), and dried over sodium sulfate. The drying agent was removed byfiltration, and the filtrate was concentrated under reduced pressure toafford 124d as a yellow solid (1.23 g, 76%). MS: [M+H]⁺ 169

Example 124e1-(2-Nitro-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)ethanone 124e

To a solution of 124d (672 mg, 4.0 mmol) in dichloromethane (20 mL) wasadded acetyl chloride (936 mg, 12.0 mmol) and K₂CO₃ (1104 mg, 8.0 mmol).The mixture was stirred overnight. It was then filtered and the filtratewas concentrated under reduced pressure. The resulting residue waspurified by silica-gel column chromatography eluting with 100:1dichloromethane/methanol to afford 124e as white solid (500 mg, 60%).MS: [M+H]⁺ 211.2

Example 124f1-(2-Amino-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)ethanone 124f

A 50-mL single-neck round-bottomed flask was purged with nitrogen andcharged with 124e (492 mg, 2.34 mmol), 10% palladium on carbon (50% wet,234 mg), and methanol (20 mL). The mixture was evacuated, charged withhydrogen gas, and stirred at room temperature for 2 h. The hydrogen wasthen evacuated and nitrogen was charged into the flask. The catalyst wasremoved by filtration through a pad of CELITE® and the filtrate wasconcentrated under reduced pressure to afford 124f (380 mg, 80%). MS:[M+H]⁺ 181.1

Example 124g3-(5-Acetyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-one124g

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 124f (270 mg, 1.5 mmol),1,4-dioxane (20 mL), Pd₂(dba)₃ (137 mg, 0.15 mmol), XantPhos (173 mg,0.30 mmol), and cesium carbonate (978 mg, 3.0 mmol). After three cyclesof vacuum/argon flush, the mixture was heated at 100° C. for 6 h. Afterthis time the reaction was cooled to room temperature. It was thenfiltered and the filtrate was evaporated in vacuo. The residue waspurified by silica-gel column chromatography eluting with 50:1dichloromethane/methanol to afford 124g (540 mg, 89%) as a yellow solid.MS: [M+H]⁺ 368.0

Example 124h3-(5-Acetyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one124h

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 124g (365 mg, 1.0 mmol),Pin₂B₂ (1.26 g, 5.0 mmol), Pd₂(dba)₃ (91 mg, 0.10 mmol), X-phos (92 mg,0.20 mmol), potassium acetate (294 mg, 3.0 mmol), and dioxane (10 mL).After three cycles of vacuum/argon flush, the mixture was heated at 60°C. for 16 h. It was then cooled to room temperature and filtered. Thefiltrate was concentrated under reduced pressure and the residue waspurified by silica-gel column chromatography eluting with 50:1 methylenechloride/methanol to afford 124h as a brown solid (330 mg, 80%). MS:[M+H]⁺ 414.2

Example 124i4-(5-(5-Acetyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)nicotinaldehyde124i

Following the procedure described in Example 123d, and starting with2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (200 mg, 0.57 mmol) and 124h (343 mg, 0.83 mmol), 124i was obtainedas a yellow solid (300 mg, 86%). MS-ESI: [M+H]⁺ 611.3

Example 1242-[4-[5-[(5-acetyl-6,7-dihydro-4H-pyrazolo[1,5-a]pyrazin-2-yl)amino]-1-methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-6-tert-butyl-8-fluoro-phthalazin-1-one124

Following the procedure in Example 120, and starting with 124i (200 mg,0.33 mmol), 124 was obtained as a white solid (54 mg, 27%). MS-ESI:[M+H]⁺ 613.3. ¹H NMR (500 MHz, DMSO-d₆, T=80° C.) δ 8.53 (d, J=8.0 Hz,1H), 8.47 (d, J=5.0 Hz, 1H), 7.94-7.92 (m, 2H), 7.84 (d, J=2.0 Hz, 1H),7.67 (dd, J=2.5, 22.0 Hz, 1H), 7.46 (d, J=8.5 Hz, 1H), 7.34 (d, J=4.0Hz, 1H), 5.98 (s, 1H), 4.63-4.57 (m, 3H), 4.44 (d, J=8.0 Hz, 2H), 3.98(bs, 2H), 3.89-3.86 (m, 2H), 3.58 (s, 3H), 2.08 (s, 3H), 1.41 (s, 9H).

Example 125a5-Bromo-1-methyl-3-(5-methyloxazol-2-ylamino)pyridin-2(1H)-one 125a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 5-methyloxazol-2-amine(276 mg, 2.82 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (753 mg, 2.82mmol), tris-(dibenzylideneacetone)dipalladium(0) (256 mg, 0.28 mmol),XantPhos (324 mg, 0.56 mmol), Cs₂CO₃ (1.8 g, 5.64 mmol), and 1,4-dioxane(30 mL). After three cycles of vacuum/argon flush, the mixture washeated at 92° C. for 3 hrs. It was then cooled to room temperature andfiltered. The filtrate was concentrated under reduced pressure and theresulting residue was purified by silica-gel column chromatographyeluting with 100:1 dichloromethane/methanol to afford 125a as a whitesolid (702 mg, 88%). MS-ESI: [M+H]⁺ 284.1.

Example 125b(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-methyloxazol-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 125b

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 125a (150 mg, 0.53mmol),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicacid 116c (438 mg, 1.06 mmol), Pd(dppf)Cl₂ (39 mg, 0.053 mmol), K₃PO₄(225 mg, 1.06 mmol), sodium acetate (87 mg, 1.06 mmol), water (0.5 mL),and acetonitrile (10 mL). After three cycles of vacuum/argon flush, themixture was heated at 90° C. for 1 h. It was then cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure and the resulting residue was purified by silica-gel columnchromatography eluting with dichloromethane/methanol (100:1 to 50:1) toafford 125b as a yellow solid (120 mg, 40%). MS-ESI: [M+H]⁺ 573.3.

Example 1256-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[(5-methyloxazol-2-yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one125

A mixture of 125b (114 mg, 0.20 mmol) and lithium hydroxide (120 mg, 5.0mmol) in i-propanol/THF/water (2:2:1, 10 mL) was stirred at 35° C. for30 mins. The mixture was concentrated under reduced pressure and theresidue was diluted with water (5 mL). The resulting mixture wasextracted with dichloromethane three times. The combined organic layerwas then concentrated under reduced pressure and the resulting residuewas purified by reverse-phase prep-HPLC to afford 125 (52 mg, 49%).MS-ESI: [M+H]⁺ 530.9. ¹H NMR (500 MHz, DMSO-d₆) δ 9.25 (s, 1H), 8.57 (d,J=5.0 Hz, 1H), 8.53 (d, J=3.0 Hz, 1H), 8.25 (d, J=2.5 Hz, 1H), 7.90 (d,J=1.0 Hz, 1H), 7.78 (dd, J=1.0, 13.0 Hz, 1H), 7.61 (d, J=2.0 Hz, 1H),7.50 (d, J=5.0 Hz, 1H), 6.64 (d, J=1.5 Hz, 1H), 4.92 (bs, 1H), 4.40 (d,J=7.0 Hz, 2H), 3.60 (s, 3H), 2.22 (s, 3H), 1.39 (s, 9H).

Example 126a 3-Cyclopropyl-3-oxopropanenitrile 126a

To a solution of acetonitrile (0.34 mL, 6.58 mmol) in THF (3 mL) at −78°C. under N₂ protection was added lithium di-i-propylamide (3.3 mL, 2M inTHF, 6.58 mmol) drop-wise. The reaction mixture was stirred at −78° C.for 3 h. Then ethyl cyclopropanecarboxylate (0.50 g, 4.38 mmol) in THF(2 mL) was added and the mixture was allowed to warm to room temperaturefor a period of 1 h. Water (2 mL) was added and the solvent was removedunder reduced pressure. Dichloromethane (2 mL) was added and the pH ofthe mixture was adjusted to 5 with 2N HCl. It was then extracted withdichloromethane (5 mL×2). The combined organic layer was dried overNa₂SO₄ and concentrated to afford 126a as a yellow oil, which was usedin the next step without further purification.

Example 126b 3-Cyclopropyl-1H-pyrazol-5-amine 126b

To a solution of 126a (477 mg, 4.38 mmol) in methanol (5 mL) was addedhydrazine hydrate (5 mL, 80%). The reaction mixture was heated at 75° C.for 15 h. The methanol was removed under reduced pressure and theresidue was extracted with dichloromethane (2×8 mL). The combinedextract was dried over Na₂SO₄ and concentrated. The residue was purifiedby flash column eluting with 100:1 dichloromethane/methanol to afford126b as a yellow oil (250 mg, 46%, over two steps). MS: [M+H]⁺ 124.

Example 126c tert-Butyl 5-Amino-3-cyclopropyl-1H-pyrazole-1-carboxylate126c

To a mixture of 126b (0.25 g, 2.0 mmol) and K₂CO₃ (0.828 g, 6.0 mmol) inTHF (5 mL) was added (Boc)₂O (0.436 g, 2.0 mmol) in THF (5 mL). Thereaction mixture was stirred at room temperature for 15 h. It was thenfiltered and the filtrate was concentrated under reduced pressure. Theresidue was purified by flash column eluting with 6:1 petroleumether/ethyl acetate to afford 126c as a white solid (240 mg, 54%). MS:[M-Boc]⁺ 124.

Example 126d5-Bromo-3-(3-cyclopropyl-1H-pyrazol-5-ylamino)-1-methylpyridin-2(1H)-one126d

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (15 mL),126c (455 mg, 1.95 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (0.40 g,1.5 mmol), and cesium carbonate (1.22 g, 3.75 mmol). After bubblingnitrogen through the resulting mixture for 30 minutes, XantPhos (87 mg,0.15 mmol) and tris(dibenzylideneacetone)dipalladium(0) (70 mg, 0.075mmol) were added. The reaction mixture was refluxed for 15 h. After thistime the reaction was cooled to room temperature and filtered. Thefiltrate was partitioned between ethyl acetate (30 mL) and water (30mL). The aqueous layer was separated and extracted with ethyl acetate(2×50 mL). The combined organic layer was washed with brine (50 mL) anddried over sodium sulfate. The drying agent was removed by filtrationand the filtrate was concentrated under reduced pressure. The residuewas purified on silica-gel column eluting with 50:1dichloromethane/methanol to afford 126d as a yellow solid (320 mg, 70%).MS: [M+H]⁺ 309. ¹H NMR (500 MHz, DMSO-d₆) δ 11.85 (s, 1H), 8.23 (s, 1H),8.02 (d, J=2.5 Hz, 1H), 7.35 (d, J=2.5 Hz, 1H), 5.77 (d, J=2.0 Hz, 1H),3.46 (s, 3H), 1.84-1.82 (m, 1H), 0.92-0.90 (m, 2H), 0.65-0.64 (m, 2H).

Example 126e5-(3-Cyclopropyl-1H-pyrazol-5-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-ylboronicAcid 126e

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 126d (205 mg, 0.665mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.0g, 4.0 mmol), dioxane (16 mL), PdCl₂(dppf) (54.3 mg, 0.066 mmol), andpotassium acetate (0.39 mg, 4.0 mmol). After bubbling argon through theresulting mixture for 30 minutes, it was stirred at 105° C. for 4 hunder argon atmosphere. It was then cooled to room temperature andfiltered. The filtrate was evaporated under reduced pressure to affordcrude 126e, which was used without further purification. MS: [M+H]⁺ 275.

Example 126f5-Bromo-3-(5-cyclopropyl-1-methyl-1H-pyrazol-3-ylamino)-1-methylpyridine-2(1H)-one126f

At 0° C., to a solution of 126e (500 mg, 1.6 mmol) in DMF (6 mL) wasadded NaH (60% in oil) (80 mg, 2.0 mmol). The reaction mixture wasstirred at 0° C. for 1 h. Iodomethane (213 mg, 1.5 mmol) was introducedand the resulting mixture was stirred at room temperature for another 2h. Then water (10 mL) was added to the mixture. The resulting suspensionwas filtered, washed with water, and dried in vacuo to afford 126f as awhite solid (350 mg, 68%). MS-ESI: [M+H]⁺ 323.1.

Example 126g(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(5-cyclopropyl-1-methyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 126g

A sealed tube equipped with a magnetic stirrer was charged with 126f(200 mg, 0.62 mmol),(2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)methylacetate 116c (268 mg, 0.65 mmol), Pd(dppf)Cl₂ (18 mg, 0.025 mmol),sodium acetate (74 mg, 0.90 mmol), K₃PO₄ (191 mg, 0.90 mmol), andacetonitrile/water (5 mL/0.5 mL). After three cycles of vacuum/argonflush, the mixture was heated at 100° C. for 2.0 h. It was then cooledto room temperature and filtered. The filtrate was evaporated underreduced pressure. The residue was purified by silica-gel columnchromatography eluting with 10:1 dichloromethane/methanol to afford 126g(100 mg, 26%) as a brown solid. MS-ESI: [M+H]⁺ 612.3.

Example 1266-tert-butyl-2-[4-[5-[(5-cyclopropyl-1-methyl-pyrazol-3-yl)amino]-1-methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one126

A mixture of 126g (100 mg, 0.16 mmol) and lithium hydroxide (72 mg, 3.0mmol) in i-propanol/THF (1:1, 4 mL) and water (1 mL) was stirred at 50°C. for 2 h. The mixture was evaporated under reduced pressure. Theresidue was purified by reverse-phase Combiflash to afford 126 (32 mg,35%) as a white solid. MS-ESI: [M+H]⁺ 570.3. ¹H NMR (500 MHz, DMSO-d₆) δ8.63 (d, J=5.0 Hz, 1H), 8.33 (d, J=2.0 Hz, 1H), 7.94 (d, J=1.5 Hz, 1H),7.56-7.55 (m, 2H), 7.53-7.51 (m, 2H), 7.33 (s, 1H), 5.53 (s, 1H)4.49-4.48 (m, 2H), 4.04-4.02 (m, 1H), 3.79 (s, 3H), 3.69 (s, 3H),1.69-1.65 (m, 1H), 1.43 (s, 9H), 0.97-0.94 (m, 2H), 0.68-0.65 (m, 2H).

Example 127a6-Chloro-2-methyl-4-(5-methyl-1H-pyrazol-3-ylamino)pyridazin-3(2H)-one127a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (20 mL),5-ethyl-1H-pyrazol-3-amine (971 mg, 10.0 mmol),4-bromo-6-chloro-2-methylpyridazin-3(2H)-one (2.46 g, 11.0 mmol), andcesium carbonate (6.52 g, 20.0 mmol). After bubbling nitrogen throughthe suspension for 10 minutes, xantphos (1.74 g, 3.0 mmol) andtris(dibenzylideneacetone)dipalladium(0) (1.37 g, 1.5 mmol) were added.The system was subjected to three cycles of vacuum/argon flush andheated at reflux for 2 h. It was then filtered immediately when thereaction mixture was still hot. The solid was washed with dioxane (3×30mL) and the combined filtrate was concentrated under reduced pressure.The residue was purified by silica-gel chromatography eluting with 6:1petroleum ether/ethyl acetate to afford 127a (1.8 g, 75%) as a yellowsolid. MS-ESI: [M+H]⁺ 239.9

Example 127b6-Chloro-4-(1,5-dimethyl-1H-pyrazol-3-ylamino)-2-methylpyridazin-3(2H)-one127b

To a mixture of 127a (480 mg, 2.0 mmol) in anhydrous DMF (10 mL) wasadded NaH (purity 60%) (64 mg, 1.6 mmol) at 0° C. and the resultingmixture was stirred for 0.5 h. To the mixture was added iodomethane (227mg, 1.6 mmol) in DMF (5 mL) dropwise at 0° C. The reaction mixture wasstirred for additional 1.5 h and quenched with water (20 mL). It wasthen extracted with dichloromethane (3×20 mL) and the combined organiclayer was evaporated under reduced pressure. The residue was purified byreverse-phase Combiflush (A: 2% aqueous NH₄HCO₃, B: acetonitrile) toafford 127b (120 mg, 24%) as a light yellow solid. MS-ESI: [M+H]⁺ 254.3

Example 127c(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(1,5-dimethyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)pyridin-3-yl)methylAcetate 127c

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 127b (120 mg, 0.47mmol,),(2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)methylacetate 116c (536 mg, 1.3 mmol), Pd(dppf)Cl₂ (34 mg, 0.047 mmol), K₃PO₄(199 mg, 0.94 mmol), sodium acetate (77 mg, 0.94 mmol), acetonitrile (10mL), and water (0.2 mL). After three cycles of vacuum/argon flush, themixture was heated at 100° C. for 2 h. It was then cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure and the resulting residue was purified by silica-gel columnchromatography eluting with dichloromethane/methanol (100:1 to 30:1) toafford 127c (180 mg, 65%) as a black oil. MS-ESI: [M+H]⁺ 587.1

Example 1276-tert-butyl-2-[4-[5-[(1,5-dimethylpyrazol-3-yl)amino]-1-methyl-6-oxo-pyridazin-3-yl]-3-(hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one127

To a solution of 127c (176 mg, 0.30 mmol) in propan-2-ol (4 mL),tetrahydrofuran (4 mL), and water (1.0 mL) was added lithium hydroxide(72 mg, 3.0 mmol). The mixture was stirred at 30° C. for 2 h. It wasthen evaporated under reduced pressure and the residue was purified byreverse-phase prep-HPLC to afford 127 (30 mg, 18%) as a white solid.MS-ESI: [M+H]⁺ 544.8. ¹H NMR (500 MHz, MeOD) δ 8.66 (d, J=5.0 Hz, 1H),8.51 (d, J=2.5 Hz, 1H), 7.88 (s, 1H), 7.88 (s, 1H), 7.76-7.72 (m, 2H),5.97 (s, 1H), 4.68 (s, 2H), 3.89 (s, 3H), 3.75 (s, 3H), 2.29 (s, 3H),1.47 (s, 9H)

Example 128a 5-Methyl-2-nitro-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine128a

A 1-L single-neck round-bottomed flask equipped with a magnetic stirrerand nitrogen inlet was charged with THF (350 mL), 124c (10.0 g, 32.2mmol), 2M methylamine solution in THF (113 mL, 225 mmol) and stirred atroom temperature for 72 h. After this time the reaction was concentratedto dryness under reduced pressure, and the resulting solid was stirredwith a mixture of ethyl acetate (75 mL) and 10% aqueous potassiumcarbonate (75 mL). The aqueous layer was separated and extracted withethyl acetate (2×75 mL). The combined organic extracts were washed with10% aqueous potassium carbonate (75 mL), followed by brine (50 mL) anddried over sodium sulfate. The drying agent was removed by filtration,and the filtrate concentrated under reduced pressure to afford 128a in97% yield (5.70 g) as a yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 6.62 (s,1H), 4.28 (t, 2H, J=5.4 Hz), 3.67 (s, 2H), 2.95 (t, 2H, J=5.4 Hz), 2.52(s, 3H); MS (ESI+) m/z 183.0 (M+H)

Example 128b 5-Methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-amine128b

A 500-mL Parr reactor bottle was purged with nitrogen and charged with10% palladium on carbon (50% wet, 800 mg dry weight) and a solution of128a (4.00 g, 2.20 mmol) in ethanol (160 mL). The bottle was attached toParr hydrogenator, evacuated, charged with hydrogen gas to a pressure of45 psi and shaken for 2 h. After this time, the hydrogen was evacuated,and nitrogen was charged into the bottle. CELITE® 521 (1.0 g) was added,and the mixture was filtered through a pad of CELITE® 521. The filtercake was washed with ethanol (2×75 mL), and the combined filtrates wereconcentrated to dryness under reduced pressure to afford a 99% yield of128b (3.31 g) as an orange solid. ¹H NMR (300 MHz, CDCl₃) δ 5.34 (s,1H), 3.98 (t, 2H, J=5.4 Hz), 3.52 (s, 3H), 2.84 (t, 2H, J=5.7 Hz), 2.45(s, 3H); MS (ESI+) m/z 153.1 (M+H)

Example 128c5-Bromo-1-methyl-3-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one128c

A sealed tube equipped with a magnetic stirrer was charged with 128b(1.02 g, 6.7 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (2.15 g, 8.1mmol), Pd₂(dba)₃ (610 mg, 0.67 mmol),2,2-bis(diphenylphosphino)-1,1-binaphthyl (775 mg, 1.34 mmol), cesiumcarbonate (4.37 g, 13.6 mmol), and 1,4-dioxane (30 mL). After threecycles of vacuum/argon flush, the mixture was heated at 110° C. for 2 h.It was then filtered and the filtrate was evaporated in vacuo. Theresidue was purified by silica gel column chromatography eluting withdichloromethane/methanol (15:1, V/V) to afford 128c (380 mg, 14%) as awhite solid. LCMS: [M+H]⁺ 338

Example 128d1-Methyl-3-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one128d

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a condenser was charged with 128c (1.0 g, 3 mmol), Pin₂B₂(3.8 g, 15 mmol), Pd(dppf)Cl₂ (137 mg, 0.15 mmol), X-phos (143 mg, 0.3mmol), potassium acetate (88 mg, 9 mmol), and 1,4-dioxane (50 mL). Afterthree cycles of vacuum/argon flush, the reaction mixture was heated at60° C. for 15 h. It was then cooled to room temperature and filtered.The filtrate was concentrated under reduced pressure and the resultingresidue was washed with petroleum ether to afford 128d as a yellow solid(0.87 g, 75%). MS: [M+H]⁺ 386

Example 128e2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde128e

A 25-mL round-bottomed flask equipped with a reflux condenser wascharged with 128d (193 mg, 0.50 mmol),2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (180 mg, 0.50 mmol),1,1′-bis(diphenylphosphino)ferrocenedichloropalladium(II) (17 mg, 0.025mmol), K₃PO₄ (212 mg, 1.0 mmol), sodium acetate (82 mg, 1.0 mmol),acetonitrile (6 mL), and water (0.5 mL). After three cycles ofvacuum/argon flush, the mixture was heated at 100° C. for 1 h. Analysisof the reaction mixture by LCMS showed completed conversion to thedesired product. The reaction mixture was cooled to room temperature andconcentrated under reduced pressure. The residue was diluted withdichloromethane (20 mL) and water (10 mL). The aqueous layer wasseparated and extracted with dichloromethane (3×10 mL). The combinedorganic layer was dried over Na₂SO₄ and filtered. The filtrate wasconcentrated under reduced pressure. The dark residue was purified bysilica-gel column chromatography eluting with dichloromethane/methanol(80:1 to 30:1) to afford 128e (190 mg, 65%) as a yellow solid. MS-ESI:[M+H]⁺ 583.3

Example 1286-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[(5-methyl-6,7-dihydro-4H-pyrazolo[1,5-a]pyrazin-2-yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one128

To a solution of 128e (158 mg, 0.27 mmol) in methanol/dichloromethane(5/5 mL) was added NaBH₄ (31 mg, 0.82 mmol) at room temperature. Afterthe reaction was stirred for 1 h, LCMS indicated the reaction wascomplete. The reaction was quenched with water (10 mL) and concentratedunder reduced pressure. The residue was extracted with dichloromethane(3×20 mL). The combined organic layer was washed with brine (30 mL),dried over Na₂SO₄, filtered, and concentrated under reduced pressure.The residue was purified by reverse-phase prep-HPLC to afford 128 (95mg, 60%) as a white solid. MS-ESI: [M+H]⁺ 585.3. ¹H NMR (500 MHz, CDCl₃)δ 8.66 (d, J=5.0 Hz, 1H), 8.35 (d, J=2.0 Hz, 1H), 8.00 (d, J=2.5 Hz,1H), 7.58-7.54 (m, overlap, 4H), 7.44 (s, 1H), 5.70 (s, 1H), 4.52-4.39(m, 2H), 4.10-4.09 (m, 3H), 3.70 (s, 3H), 3.59 (s, 2H), 2.88-2.86 (m,2H), 2.47 (s, 3H), 1.43 (s, 9H).

Example 129a5-Bromo-1-methyl-3-(5-methylisoxazol-3-ylamino)pyridin-2(1H)-one 129a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 5-methylisoxazol-3-amine(1.0 g, 10.2 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (4.09 g, 15.3mmol), Pd₂(dba)₃ (467 mg, 0.51 mmol), Xantphos (598 mg, 1.02 mmol),Cs₂CO₃ (6.65 g, 20.4 mmol), and dioxane (50 mL). After three cycles ofvacuum/argon flush, the reaction mixture was heated at 100° C. for 3 h.Analysis of the reaction mixture by LCMS showed completed conversion tothe desired product. It was filtered when the mixture was still hot. Thefiltrate was cooled to room temperature and the resulting precipitatewas collected by filtration to afford 129a (1.6 g, 55%) as a yellowsolid. MS-ESI: [M+H]⁺ 284.1

Example 129b(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-methylisoxazol-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 129b

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicacid 116c (438 mg, 1.06 mmol), 129a (150 mg, 0.60 mmol), Pd(dppf)Cl₂ (19mg, 0.026 mmol), K₃PO₄ (224 mg, 1.06 mmol), sodium acetate (87 mg, 1.06mmol), water (5 drops), and acetonitrile (10 mL). After three cycles ofvacuum/argon flush, the reaction mixture was heated at 100° C. for 1 h.Analysis of the reaction mixture by LCMS showed complete conversion tothe desired product. The mixture was cooled down to room temperature andfiltered. The filtrate was concentrated under reduced pressure to afford129b (300 mg, 87%) as a dark oil, which was used in next step withoutfurther purification. MS-ESI: [M+H]⁺ 573.3

Example 1296-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[(5-methylisoxazol-3-yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one129

To a solution of 129b (280 mg, 0.49 mmol) in THF (4 mL), i-propanol (4mL), and water (2 mL) was added lithium hydroxide (24 mg, 0.98 mmol).The reaction mixture was stirred at room temperature for 1 h. It wasconcentrated under reduced pressure and the residue was purified byreverse-phase prep-HPLC to afford 129 as a white solid (85 mg, 33%).MS-ESI: [M+H]⁺ 531.3. ¹H NMR (500 MHz, DMSO-d₆) δ 9.03 (s, 1H), 8.58 (d,J=5.0 Hz, 1H), 8.54 (d, J=3.0 Hz, 1H), 7.99 (d, J=2.0 Hz, 1H), 7.91 (d,J=1.0 Hz, 1H), 7.78 (dd, J=1.0, 13.0 Hz, 1H), 7.56 (d, J=2.0 Hz, 1H),7.51 (d, J=5.0 Hz, 1H), 6.26 (s, 1H), 4.92 (s, 1H), 4.43 (d, J=6.0 Hz,2H), 3.61 (s, 3H), 2.32 (s, 3H), 1.40 (s, 9H).

Example 130a5-Bromo-1-methyl-3-(1-methyl-1H-imidazol-4-ylamino)pyridin-2(1H)-one130a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (50 mL),1-methyl-1H-imidazol-4-amine (1.1 g, 11.3 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (3.0 g, 11.3 mmol), Pd₂(dba)₃ (1.0g, 1.13 mmol), XantPhos (1.3 g, 2.26 mmol), and cesium carbonate (7.3 g,22.6 mmol). After three cycles of vacuum/argon flush, the mixture washeated at 92° C. for 4.5 hrs. It was then cooled to room temperature andfiltered. The filtrate was concentrated under reduced pressure and theresulting residue was purified by silica-gel column chromatographyeluting with dichloromethane/methanol (100:1 to 50:1) to afford 130a(2.4 g, 76%) as a yellow solid. MS-ESI: [M+H]⁺ 283.1

Example 130b(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(1-methyl-1H-imidazol-4-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 130b

A 50-mL round-bottomed flask equipped with a magnetic stirrer wascharged with 130a (150 mg, 0.53 mmol),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicacid 116c (438 mg, 1.06 mmol), PdCl₂(dppf) (43 mg, 0.053 mmol), K₃PO₄(225 mg, 1.06 mmol), sodium acetate (87 mg, 1.06 mmol), acetonitrile (10mL), and water (0.2 mL). After bubbling nitrogen into the mixture for 10minutes, a reflux condenser was attached to the flask and the reactionmixture was heated at 90° C. for 2.5 h. It was then cooled to roomtemperature and filtered. The filtrate was evaporated under reducedpressure and the resulting residue was purified by silica-gel columnchromatography eluting with dichloromethane/methanol (50:1 to 20:1) toafford 130b as a yellow solid (120 mg, 40%). MS-ESI: [M+H]⁺ 572.3.

Example 1306-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[(1-methylimidazol-4-yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one130

A mixture of 130b (100 mg, 0.18 mmol) and lithium hydroxide hydrate (189mg, 4.5 mmol) in i-propanol/THF/water (2:2:1, 10 mL) was stirred at 35°C. for 30 min. The mixture was evaporated under reduced pressure and theresidue was added water (5 mL). It was then extracted withdichloromethane (3×10 mL). The combined extract was concentrated underreduced pressure and the residue was purified by reverse-phase prep-HPLCto afford 130 (41.1 mg, 44.4%) as white solid. MS-ESI: [M+H]⁺ 530.3. ¹HNMR (500 MHz, DMSO-d₆) δ 8.56-8.53 (m, 2H), 7.90 (d, J=2.0 Hz, 1H), 7.78(dd, J=2.0, 12.5 Hz, 1H), 7.61 (s, 1H), 7.51 (d, J=5.0 Hz, 1H),7.41-7.39 (m, 2H), 7.37 (d, J=2.0 Hz, 1H), 6.97 (d, J=1.5 Hz, 1H),5.04-5.02 (m, 1H), 4.41-4.39 (m, 2H), 3.59 (s, 3H), 3.58 (s, 3H), 1.39(s, 9H).

Example 131a 2-Nitro-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazine 131a

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer and reflux condenser was charged with1-(2-bromoethyl)-5-(bromomethyl)-3-nitro-1H-pyrazole 124c (3.00 g, 9.59mmol) and 4M aqueous hydrobromic acid (120 mL), and the resultingmixture was heated at reflux for 24 h. After this time, the reactionmixture was concentrated under reduced pressure to approximately 6 mLvolume, and the residue was stirred in 2M aqueous sodium hydroxide (40mL) for 2 h. After this time methylene chloride was added (40 mL) andthe mixture was stirred for 15 min. The aqueous layer was separated andextracted with methylene chloride (2×50 mL). The combined organicextracts were washed with brine (100 mL) and dried over sodium sulfate.The drying agent was removed by filtration and the filtrate concentratedunder reduced pressure to afford a 62% yield (1.01 g) of 131a as a whitesolid: mp 110-112° C.; ¹H NMR (300 MHz, CDCl₃) 6.68 (s, 1H), 4.87 (s,2H), 4.28 (t, 2H, J=5.4 Hz), 4.20 (t, 2H, J=5.1 Hz); MS (ESI+) m/z 170.0(M+H).

Example 131b 6,7-Dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-amine 131b

A 500-mL Parr hydrogenation bottle was purged with nitrogen and chargedwith 131a (1.01 g, 5.92 mmol), 10% palladium on carbon (50% wet, 125 mgdry weight) and ethanol (50 mL). The bottle was evacuated, charged withhydrogen gas to a pressure of 25 psi and shaken for 2 h on a Parrhydrogenation apparatus. The hydrogen was then evacuated and nitrogencharged to the bottle. The catalyst was removed by filtration through apad of CELITE® 521 and the filtrate concentrated under reduced pressure.The resulting residue was purified by column chromatography using 400 ccof silica gel and eluting with 3% methanol in methylene chloride. Thefractions were collected to afford, after concentrating under reducedpressure, a 73% yield (601 mg) of 131b as a yellow solid: mp 74-76° C.¹H NMR (300 MHz, CDCl₃) δ 5.37 (s, 1H), 4.72 (s, 2H), 4.07 (t, 2H, J=5.1Hz), 3.98 (t, 2H, J=5.1 Hz), 3.57 (br s, 2H); MS (ESI+) m/z 140.4 (M+H).

Example 131c5-Bromo-3-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methylpyridin-2(1H)-one131c

A 50-mL three-neck round-bottomed flask equipped with a magneticstirrer, reflux condenser and nitrogen inlet was charged with1,4-dioxane (20 mL), 131b (600 mg, 4.31 mmol),3,5-dibromo-1-methylpyridine-2(1H)-one (1.44 g, 5.40 mmol) and cesiumcarbonate (3.08 g, 9.48 mmol). After bubbling nitrogen through theresulting solution for 30 min, Xantphos (300 mg, 0.52 mmol) andtris(dibenzylideneacetone)dipalladium(0) (320 mg, 0.35 mmol) were added,and the reaction mixture was heated at reflux for 2 h. After this timethe reaction was cooled to room temperature, partitioned between ethylacetate (75 mL) and water (75 mL) and filtered. The aqueous layer wasseparated and extracted with ethyl acetate (2×25 mL). The organic layerswere combined and washed with brine (50 mL) and dried over sodiumsulfate. The drying agent was removed by filtration and the filtrateconcentrated under reduced pressure. The resulting residue was purifiedby column chromatography using 500 cc of silica gel and eluting with 1%methanol in methylene chloride. The fractions were collected to afford,after concentrating under reduced pressure, a 31% yield (433 mg) of 131cas a green solid: mp 195-197° C.; ¹H NMR (300 MHz, CDCl₃) 7.92 (d, 1H,J=2.4 Hz), 7.44 (s, 1H), 6.90 (d, 1H, J=2.4 Hz), 5.65 (s, 1H), 4.80 (s,2H), 4.13 (s, 2H), 3.61 (s, 5H); MS (ESI+) m/z 324.9 (M+H).

Example 131d3-(6,7-Dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one131d

A 250-mL round bottomed flask equipped with a magnetic stirrer and areflux condenser was charged with the mixture of 131c (1.3 g, 4.0 mmol),bis(pinacolato)diboron (2.03 g, 8.0 mmol), PdCl₂(dppf) (439 mg, 0.60mmol), potassium acetate (784 mg, 8.0 mmol), and 1,4-dioxane (60 mL).After bubbling nitrogen through the mixture for 30 minutes, it washeated at reflux for 15 h. The mixture was cooled to room temperatureupon completion of the reaction and filtered. The solid was washed withethyl acetate (100 mL). The combined filtrate was evaporated underreduced pressure and the residue was purified by silica-gel columnchromatography eluting with 30:1 dichloromethane/methanol to afford 131d(446 mg, 30%). MS: [M+H]⁺ 373.

Example 131e2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde131e

A 50-mL round-bottomed flask equipped with a reflux condenser wascharged with2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (216 mg, 1 eq., 0.60 mmol), 131d (446 mg, 2 eq., 1.2 mmol),Pd₂(dba)₃ (55 mg, 0.1 eq., 0.060 mmol), Cy₃P (67 mg, 0.4 eq., 0.24mmol), Cs₂CO₃ (395 mg, 2 eq., 1.2 mmol), water (0.5 mL), and dioxane (10mL). After three cycles of vacuum/N₂ flush, the reaction mixture wasstirred at 100° C. for 2 hrs. It was then cooled to room temperature andfiltered. The filtrate was evaporated under reduced pressure and theresulting residue was purified by silica-gel column chromatographyeluting with 4:1 petroleum ether/ethyl acetate to afford 131e (272 mg,80%) as a brown solid. MS-ESI: [M+H]⁺ 569.8

Example 1316-tert-butyl-2-[4-[5-(6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one131

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with 131e (220 mg, 1.0 eq., 0.39 mmol), NaBH₄ (73mg, 5.0 eq., 1.90 mmol), and methanol (10 mL). The mixture was stirredat room temperature for 1 h and quenched with water (5 mL). It was thenconcentrated under reduced pressure and resulting residue was extractedwith dichloromethane (3×10 mL). The combined organic layer wasconcentrated under reduced pressure and the residue was purified byreverse-phase prep-HPLC to afford 131 (105 mg, 47%). MS-ESI: [M+H]⁺571.8. ¹H NMR (500 MHz, CDCl₃) δ 8.66 (d, J=5.0 Hz, 1H), 8.35 (d, J=2.5Hz, 1H), 8.03 (d, J=2.5 Hz, 1H), 7.59-7.53 (m, 4H), 7.48 (s, 1H), 5.73(s, 1H), 4.81 (s, 2H), 4.51 (bs, 2H), 4.12-4.05 (m, overlap, 5H), 3.73(s, 3H), 1.46 (s, 9H).

Example 132a (R)-(6-Aminopyridin-3-yl)(3-methylmorpholino)methanone 132a

To a solution of (R)-3-methylmorpholine (2.02 g, 20 mmol) in ethanol (25mL) was added 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI)(3.33 g, 17.4 mmol), hydroxybenzotriazole (HOBt) (2.35 g, 17.4 mmol),and 6-aminonicotinic acid (2.0 g, 14.5 mmol). After stirring for 18 h atroom temperature, the reaction suspension was filtered and the filtratewas concentrated under reduced pressure. The residue was purified bysilica-gel column chromatography eluting with 3:1 ethylacetate/petroleum ether to afford 132a as a white solid (1.6 g, 36%).MS-ESI: [M+H]⁺ 222.3.

Example 132b(R)-5-Bromo-1-methyl-3-(5-(3-methylmorpholine-4-carbonyl)pyridin-2-ylamino)pyridin-2(1H)-one132b

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 132a (332 mg, 1.5 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (H-001) (480 mg, 1.8 mmol), andcesium carbonate (978 mg, 3.0 mmol). After bubbling nitrogen through thesuspension for 3 minutes, Xantphos (87 mg, 0.15 mmol) andtris(dibenzylideneacetone)dipalladium(0) (69 mg, 0.075 mmol) were added.The system was subjected to three cycles of vacuum/argon flush andheated at reflux for 2.5 h. It was then cooled to room temperature andfiltered. The solid was washed with dichloromethane (2×50 mL) and thecombined filtrate was concentrated under reduced pressure. The residuewas purified by silica-gel column chromatography eluting with petroleumether/ethyl acetate (2:1 to 1:2) to afford 132b (430 mg, 70%) as ayellow solid. MS-ESI: [M+H]⁺ 407.3

Example 132c(R)-1-Methyl-5-(5-(3-methylmorpholine-4-carbonyl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-ylboronicAcid 132c

A 100-mL round-bottomed flask equipped with a reflux condenser wascharged with 132b (580 mg. 1.42 mmol), bis(pinacolato)diboron (1.2 g,4.5 mmol), Pd₂(dba)₃ (137 mg, 0.15 mmol), X-phos (71 mg, 0.15 mmol),potassium acetate (294 mg, 3.0 mmol), and 1,4-dioxane (20 mL). Afterthree cycles of vacuum/argon flush, the mixture was heated at 70° C. for2 h. Then it was filtered and the filtrate was evaporated under reducedpressure. The residue was washed with petroleum ether to afford 132c(500 mg, 94%) as a white solid, which was used in the next step withoutfurther purification. MS-ESI: [M+H]⁺ 373.1

Example 132d(R)-2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-(3-methylmorpholine-4-carbonyl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde132d

A 25-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 132c (260 mg, 0.70mmol),2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotin-aldehyde103b (180 mg, 0.50 mmol), K₃PO₄ (297 mg, 1.4 mmol), sodium acetate (114mg, 1.4 mmol), 1,1′-bis(diphenylphosphino)ferrocenedichloropalladium(II)(57 mg, 0.070 mmol), and acetonitrile/water (10/0.2 mL). After threecycles of vacuum/N₂ flush, the mixture was heated at 100° C. 1.5 h.Analysis of the reaction mixture by LCMS showed complete conversion tothe desired product. The reaction mixture was cooled to room temperatureand concentrated under reduced pressure. The residue was diluted withdichloromethane (20 mL) and water (20 mL). The aqueous layer wasseparated and extracted with dichloromethane (3×20 mL). The combinedorganic layer was dried over Na₂SO₄, filtered, and concentrated underreduced pressure. The dark residue was purified by silica-gel columnchromatography eluting with 60:1 dichloromethane/methanol to afford 132d(120 mg, 37%) as a yellow solid. MS-ESI: [M+H]⁺ 652.3

Example 1326-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[[5-[(3R)-3-methylmorpholine-4-carbonyl]-2-pyridyl]amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one132

A solution of 132d (120 mg, 0.18 mmol) in methanol/dichloromethane (4/4mL) was added NaBH₄ (15 mg, 0.60 mmol). The mixture was stirred at roomtemperature for 1 h and quenched with water (2 mL). It was thenevaporated under reduced pressure and the residue was purified byreverse-phase prep-HPLC to afford 132 (60 mg, 50%) as a white solid.MS-ESI: [M+H]⁺ 654.3. ¹H NMR (500 MHz, MeOD-d₄) δ 8.94 (d, J=2.5 Hz,1H), 8.61 (d, J=5.0 Hz, 1H), 8.51 (d, J=2.5 Hz, 1H), 8.35 (d, J=2.0 Hz,1H) 7.88 (d, J=1.5 Hz, 1H), 7.75 (d, J=1.5 Hz, 1H), 7.72-7.69 (m, 1H),7.65 (d, J=5.0 Hz, 1H), 7.61 (d, J=2.5 Hz, 1H), 7.13 (d, J=7.5 Hz, 1H),4.62-4.58 (m, 2H), 4.36-4.31 (m, 1H), 3.61-3.84 (m, 2H), 3.71-3.67 (m,overlap, 5H), 3.56-3.47 (m, 2H), 1.47 (s, 9H), 1.39 (d, J=7.0 Hz, 3H).

Example 133a5-(2-Methoxyethyl)-2-nitro-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine133a

To a solution of 2-nitro-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine 124d(190 mg, 1.13 mmol) in acetonitrile (10 mL) was added K₂CO₃ (311.9 mg,2.26 mmol) and 1-bromo-2-methoxyethane (188.3 mg, 1.36 mmol). Thereaction mixture was heated at 80° C. for 17 h under microwaveirradiation. Analysis of the reaction mixture by LCMS showed completeconversion to the desired product. The mixture was cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure to afford 133a as a white solid (230 mg, 90%), which was usedin the next step without further purification. MS-ESI: [M+H]⁺ 227.0

Example 133b5-(2-Methoxyethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-amine 133b

To a solution of 133a (286 mg, 1.26 mmol) in methanol (10 mL) was addedPd/C (28.6 mg). The system was evacuated and then refilled with H₂.After stirring at room temperature for 2 h, the mixture was filteredoff. The filtrate was concentrated under reduced pressure to afford 133bas a yellow solid (240 mg, 97%), which was used in the next step withoutfurther purification. MS-ESI: [M+H]⁺ 197.0

Example 133c5-Bromo-3-(5-(2-methoxyethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methylpyridin-2(1H)-one133c

A 100-mL round-bottomed flask equipped with a magnetic stirrer and areflux condenser was charged with 133b (230 mg, 1.17 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (468.4 mg, 1.76 mmol), Pd₂(dba)₃(53.5 mg, 0.0585 mmol), Xantphos (67.6 mg, 0.117 mmol), Cs₂CO₃ (762.8mg, 2.34 mmol), and dioxane (20 mL). After three cycles of vacuum/N₂flush, the mixture was heated at 100° C. for 3 h. Analysis of thereaction mixture by LCMS showed complete conversion to the desiredproduct. It was cooled to room temperature and filtered. The filtratewas concentrated under reduced pressure. The residue was purified bysilica-gel column chromatography eluting with 40:1dichloromethane/methanol to afford 133c as a dark solid (380 mg, 85%).MS-ESI: [M+H]⁺ 382.2

Example 133d3-(5-(2-Methoxyethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one133d

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 133c (330 mg, 0.86mmol), Pin₂B₂ (329 mg, 1.30 mmol), Pd₂(dba)₃ (40 mg, 0.043 mmol), X-phos(41 mg, 0.086 mmol), potassium acetate (169 mg, 1.726 mmol), and dioxane(10 mL). After three cycles of vacuum/N₂ flush, the mixture was heatedat 70° C. for 2 h. Analysis of the reaction mixture by LCMS showedcomplete conversion to the desired product. It was cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure. The residue was washed with petroleum ether to afford 133d asa dark oil (240 mg, 80%), which was used in the next step withoutfurther purification. MS-ESI: [M+H]⁺ 348.3

Example 133e2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(5-(2-methoxyethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde133e

A 25-mL round-bottomed flask equipped with a reflux condenser wascharged with2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (100 mg, 0.28 mmol), 133d (144.4 mg, 0.42 mmol), Pd(dppf)Cl₂ (11.3mg, 0.0139 mmol), K₃PO₄ (117.8 mg, 0.556 mmol), sodium acetate (45.6 mg,0.556 mmol), acetonitrile (10 mL), and water (3 drops). After threecycles of vacuum/N₂ flush, the mixture was heated at 100° C. for 1 hunder N₂ protection. Analysis of the reaction mixture by LCMS showedcomplete conversion to the desired product. It was cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure. The residue was purified by silica-gel column chromatographyeluting with 40:1 dichloromethane/methanol to afford 133e as a yellowsolid (140 mg, 80%). MS-ESI: [M+H]⁺ 627.3

Example 1336-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[5-[[5-(2-methoxyethyl)-6,7-dihydro-4H-pyrazolo[1,5-a]pyrazin-2-yl]amino]-1-methyl-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one133

To a solution of 133e (180 mg, 0.287 mmol) in dichloromethane (5 mL) andmethanol (5 mL) was added NaBH₄ (21.7 mg, 0.574 mmol). After stirring atroom temperature for 1 h, it was quenched with aqueous NH₄Cl (5 mL) andconcentrated under reduced pressure. The residue was extracted withdichloromethane (3×20 mL) The combined organic layer was washed withbrine, dried over Na₂SO₄, concentrated under reduced pressure, andpurified by reverse-phase prep-HPLC to afford 133 (50 mg, 27%) as awhite solid. MS-ESI: [M+H]⁺ 629.3. ¹H NMR (500 MHz, DMSO-d₆) δ 8.57 (d,J=5.5 Hz, 1H), 8.54 (d, J=2.5 Hz, 1H), 8.22 (s, 1H), 8.04 (d, J=2.5 Hz,1H), 7.90 (d, J=1.5 Hz, 1H), 7.78 (dd, J=1.5, 13.5 Hz, 1H), 7.51 (d,J=5.5 Hz, 1H), 7.39 (d, J=2.0 Hz, 1H), 5.89 (s, 1H), 4.90-4.88 (m, 1H),4.42 (s, 2H), 3.92 (t, J=5.5 Hz, 2H), 3.61 (s, 2H), 3.59 (s, 3H), 3.50(t, J=5.0 Hz, 2H), 3.25 (s, 3H), 2.90 (t, J=5.5 Hz, 2H), 2.67 (t, J=5.0Hz, 2H), 1.40 (s, 9H).

Example 134a2-Nitro-5-(2,2,2-trifluoroethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine134a

A sealed tube equipped with a magnetic stirrer was charged with1-(2-bromoethyl)-5-(bromomethyl)-3-nitro-1H-pyrazole 124c (632 mg, 2.0mmol), 2,2,2-trifluoroethanamine (594 mg, 6.0 mmol), and DMSO (5 mL),and heated at 120° C. for 2 h. The mixture was cooled to roomtemperature and diluted with water (20 mL). The resulting mixture wasextracted with ethyl acetate (3×20 mL). The combiner organic layer wasdried and filtered. The filtrate was concentrated under reduced pressureand the residue was purified by silical-gel column chromatographyeluting with 50:1 dichloromethane/methanol to afford 134a (392 mg, 78%)as a yellow solid. MS-ESI: [M+H]⁺ 250.9

Example 134b5-(2,2,2-Trifluoroethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-amine134b

To a solution of 134a (390 mg, 1.56 mmol) in ethanol (20 mL) was addedPd/C (about 200 mg). The reaction was charged with hydrogen gas (viaballoon) and stirred at room temperature for 2 h. After reaction wascomplete, the mixture was filtered through a plug of CELITE®. Thefiltrate was concentrated under reduced pressure to afford 134b as ayellow solid (308 mg, 90%), which was used in the next step withoutfurther purification. MS-ESI: [M+H]⁺ 221.1

Example 134c5-Bromo-1-methyl-3-(5-(2,2,2-trifluoroethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one134c

A 100-mL round-bottomed flask equipped with a reflux condenser wascharged with 134b (300 mg, 1.36 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (364 mg, 1.36 mmol), cesiumcarbonate (887 mg, 2.7 mmol), and 1,4-dioxane (20 mL). After bubblingnitrogen through the suspension for 10 minutes, xantphos (78 mg, 0.136mmol) and Pd₂(dba)₃ (62 mg, 0.068 mmol) were added. The system wassubjected to three cycles of vacuum/argon flush and heated at reflux for5 h. It was then cooled to room temperature and filtered. The solid waswashed with dichloromethane (30 mL). The combined filtrate wasconcentrated under reduced pressure. The solid residue was purified bysilica-gel column chromatography eluting with dichloromethane/methanol(80/1 to 30/1) to afford 134c (420 mg, 76%) as a yellow solid. MS-ESI:[M+H]⁺ 406.0

Example 134d1-Methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(5-(2,2,2-trifluoro-ethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridin-2(1H)-one134d

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 134c (400 mg, 0.98mmol), Pin₂B₂ (750 mg, 2.96 mmol), Pd₂(dba)₃ (45 mg, 0.05 mmol), x-phos(48 mg, 0.1 mmol), potassium acetate (294 mg, 3.0 mmol), and 1,4-dioxane(15 mL). The reaction mixture was subjected to three cycles ofvacuum/argon flush and heated at 65° C. for 3 h. It was then cooled toroom temperature and filtered. The filtrate was concentrated underreduced pressure. The resulting residue was washed with petroleum etherto afford 134d (400 mg, 90%) as a brown solid, which was used in thenext step without further purification. MS-ESI: [M+H]⁺ 453.9

Example 134e2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-6-oxo-5-(5-(2,2,2-trifluoroethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1,6-dihydropyridin-3-yl)nicotinaldehyde134e

A 25-mL round-bottomed flask equipped with a reflux condenser wascharged with 134d (200 mg, 0.44 mmol),2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (159 mg, 0.44 mmol), K₃PO₄ (186 mg, 0.88 mmol), sodium acetate (72mg, 0.88 mmol),1,1′-bis(diphenylphosphino)ferrocenedichloropalladium(II) (16 mg, 0.022mmol), and acetonitrile/water (8/0.2 mL). After three cycles ofvacuum/N₂ flush, the mixture was heated at 100° C. for 1 h under N₂protection. Analysis of the reaction mixture by LCMS showed completeconversion to the desired product. The reaction mixture was cooled toroom temperature and diluted with dichloromethane (30 mL) and water (15mL). The water layer was separated and extracted with dichloromethane(2×15 mL). The combined organic extract was dried over Na₂SO₄, filtered,and concentrated under reduced pressure. The dark residue was purifiedby silica-gel column chromatography eluting withdichloromethane/methanol (80/1 to 30/1) to afford 134e (128 mg, 45%) asa yellow solid. MS-ESI: [M+H]⁺ 651.3

Example 1346-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-6-oxo-5-[[5-(2,2,2-trifluoroethyl)-6,7-dihydro-4H-pyrazolo[1,5-a]pyrazin-2-yl]amino]-3-pyridyl]-2-pyridyl]phthalazin-1-one134

To a solution of 134e (110 mg, 0.169 mmol) inmethanol/dichloromethane(4/4 mL) was added NaBH₄ (19 mg, 0.51 mmol) atroom temperature. After the reaction was stirred for 1 h, LCMS indicatedthe reaction was complete. The mixture was quenched with water (20 mL)and extracted with dichloromethane (3×30 mL). The combined organic layerwas washed with brine (20 mL), dried over Na₂SO₄, filtered, andconcentrated under reduced pressure. The residue was purified byreverse-phase prep-HPLC to afford 134 (50 mg, 45%) as a white solid.MS-ESI: [M+H]⁺ 653.3. ¹H NMR (500 MHz, CDCl₃) δ 8.66 (d, J=5.0 Hz, 1H),8.35 (d, J=2.5 Hz s, 1H), 8.00 (d, J=2.5 Hz, 1H), 7.59-7.58 (m, 1H),7.56-7.55 (m, 1H), 7.53-7.52 (m, 2H), 7.45 (s, 1H), 5.73 (s, 1H),4.51-4.49 (m, 2H), 4.11-4.08 (m, 2H), 4.05-4.03 (m, 1H), 3.94 (s, 2H),3.73 (s, 3H), 3.24-3.18 (m, 4H), 1.45 (s, 9H).

Example 135a5-(2,2-Difluoroethyl)-2-nitro-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine135a

A sealed tube equipped with a magnetic stirrer was charged with1-(2-bromoethyl)-5-(bromomethyl)-3-nitro-1H-pyrazole 124c (632 mg, 2.0mmol), 2,2-difluoroethanamine (486 mg, 6.0 mmol), and DMSO (5 mL). Itwas heated at 120° C. for 2 h. The mixture was cooled to roomtemperature and diluted with water (20 mL). The resulting mixture wasextracted with ethyl acetate (3×20 mL). The combiner organic layer wasdried and filtered. The filtrate was concentrated under reduced pressureand the residue was purified by silica-gel column chromatography elutingwith 50:1 dichloromethane/methanol to afford 135a (371 mg, 80%) as ayellow solid. MS-ESI: [M+H]⁺ 233.2

Example 135b5-(2,2-Difluoroethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-amine135b

A solution of 135a (370 mg, 1.59 mmol) in ethanol (20 mL) was added Pd/C(about 200 mg). The reaction was charged with hydrogen gas (via balloon)and stirred at room temperature for 2 h. After reaction was complete,the mixture was filtered through a plug of CELITE®. The filtrate wasconcentrated reduced pressure to afford 135b as a yellow solid (293 mg,91%), which was used in the next step without further purification.MS-ESI: [M+H]⁺ 203.2

Example 135c5-Bromo-3-(5-(2,2-difluoroethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methylpyridin-2(1H)-one135c

A 50-mL round-bottomed flask equipped with a reflux condenser wascharged with 135b (290 mg, 1.43 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (383 mg, 1.43 mmol), cesiumcarbonate (932 mg, 2.86 mmol), and 1,4-dioxane (20 mL). After bubblingnitrogen through the suspension for 10 minutes, xantphos (82 mg, 0.143mmol) and Pd₂(dba)₃ (65 mg, 0.072 mmol) were added. The system wassubjected to three cycles of vacuum/argon flush and heated at reflux for5 h. It was then cooled to room temperature and filtered. The solid waswashed with dichloromethane (30 mL). The combined organic filtrate wasconcentrated under reduced pressure. The residue was purified bysilica-gel column chromatography eluting with dichloromethane/methanol(80/1 to 30/1) to afford 135c (400 mg, 72%) as a yellow solid. MS-ESI:[M+H]⁺ 387.8

Example 135d3-(5-(2,2-Difluoroethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one135d

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 135c (400 mg, 1.03mmol), Pin₂B₂ (785 mg, 3.1 mmol), Pd₂(dba)₃ (45 mg, 0.050 mmol), X-phos(48 mg, 0.10 mmol), potassium acetate (294 mg, 3.0 mmol), and1,4-dioxane (20 mL). The reaction mixture was subjected to three cyclesof vacuum/argon flush and heated at 65° C. for 3 h. It was then cooledto room temperature and filtered. The filtrate was concentrated underreduced pressure. The resulting residue was washed with petroleum etherto afford 135d (412 mg, 92%) as a brown solid, which was used in nextstep without further purification. MS-ESI: [M+H]⁺ 436.0

Example 135e2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(5-(2,2-difluoroethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde135e

A 25-mL round-bottomed flask equipped with a reflux condenser wascharged with 135d (200 mg, 0.46 mmol),2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (166 mg, 0.46 mmol), K₃PO₄ (195 mg, 0.92 mmol), sodium acetate (75mg, 0.92 mmol),1,1′-bis(diphenylphosphino)ferrocenedichloropalladium(II) (17 mg, 0.023mmol), and acetonitrile/water (8/0.2 mL). After three cycles ofvacuum/N₂ flush, the mixture was heated at 100° C. for 1 h. Analysis ofthe reaction mixture by LCMS showed complete conversion to the desiredproduct. The reaction mixture was cooled to room temperature and dilutedwith dichloromethane (30 mL) and water (15 mL). The water layer wasextracted with dichloromethane (2×15 mL). The combined organic extractwas dried over Na₂SO₄, filtered, and concentrated under reducedpressure. The dark residue was purified by silica-gel columnchromatography eluting with dichloromethane/methanol (80/1 to 30/1) toafford 135e (110 mg, 38%) as a yellow solid. MS-ESI: [M+H]⁺ 633.3

Example 1356-tert-butyl-2-[4-[5-[[5-(2,2-difluoroethyl)-6,7-dihydro-4H-pyrazolo[1,5-a]pyrazin-2-yl]amino]-1-methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one135

To a solution of 135e (100 mg, 0.158 mmol) inmethanol/dichloromethane(4/4 mL) was added NaBH₄ (18 mg, 0.475 mmol) atroom temperature. After the reaction was stirred for 1 h, LCMS indicatedthe reaction was complete. The reaction mixture was quenched with water(20 mL) and extracted with dichloromethane (3×20 mL). The combinedorganic layer was washed with brine (20 mL), dried over Na₂SO₄,filtered, and concentrated under reduced pressure. The residue waspurified by reverse-phase prep-HPLC to afford 135 (60 mg, 60%) as awhite solid. MS-ESI: [M+H]⁺ 635.3. ¹H NMR (500 MHz, CDCl₃) δ 8.66 (d,J=5.0 Hz, 1H), 8.35 (d, J=2.5 Hz, 1H), 8.00 (d, J=2.5 Hz, 1H), 7.59-7.58(m, 1H), 7.56-7.55 (m, 1H), 7.53-7.52 (m, 2H), 7.45 (s, 1H), 6.07-5.83(m, 1H), 5.72 (s, 1H), 4.51-4.49 (m, 2H), 4.11-4.08 (m, 2H), 4.05-4.03(m, 1H), 3.94 (s, 2H), 3.73 (s, 3H), 3.13-3.10 (m, 2H), 3.00-2.94 (m,2H), 1.45 (s, 9H).

Example 136a (S)-(6-Aminopyridin-3-yl)(3-methylmorpholino)methanone 136a

To a solution of (S)-3-methylmorpholine (1.5 g, 15.0 mmol) in ethanol(20 mL) was added EDCI (3.33 g, 17.4 mmol), HOBt (2.35 g, 17.4 mmol),and 6-aminonicotinic acid (2.07 g, 15.0 mmol) at room temperature. Afterstirring for 18 h, the resulting suspension was filtered. The solid waspurified by silica-gel column chromatography eluting with 2:1 petroleumether/ethyl acetate to straight ethyl acetate to afford 136a (1.0 g,30%) as a white solid. MS-ESI: 222.3 (M+H)⁺.

Example 136b(S)-5-Bromo-1-methyl-3-(5-(3-methylmorpholine-4-carbonyl)pyridine-2-ylamino)pyridin-2(1H)-one136b

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 136a (222 mg, 1.0 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (320 mg, 1.2 mmol), cesiumcarbonate (652 mg, 2 mmol), and 1,4-dioxane (10 mL). After bubblingnitrogen through the suspension for 10 minutes, Xantphos (58 mg, 0.10mmol) and tris(dibenzylideneacetone)dipalladium(0) (46 mg, 0.050 mmol)were added. The system was subjected to three cycles of vacuum/argonflush and heated at reflux for 2.5 h. It was then cooled to roomtemperature and filtered. The solid was washed with dichloromethane(2×30 mL) and the combined filtrate was concentrated under reducedpressure. The residue was purified by silica-gel column chromatographyeluting with petroleum ether/ethyl acetate (2:1 to 1:2) to afford 136b(280 mg, 69%) as a yellow solid. MS-ESI: [M+H]⁺ 407.3

Example 136c(S)-1-Methyl-5-(5-(3-methylmorpholine-4-carbonyl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-ylboronicAcid 136c

A 100-mL round-bottomed flask equipped with a reflux condenser wascharged with 136b (600 mg, 1.5 mmol), bis(pinacolato)diboron (1.2 g, 4.5mmol), Pd₂(dba)₃ (137 mg, 0.15 mmol), X-phos (71 mg, 0.15 mmol),potassium acetate (294 mg, 3.0 mmol), and 1,4-dioxane (20 mL). Afterthree cycles of vacuum/argon flush, the mixture was heated at 70° C. for2 h. It was then filtered and the filtrate was evaporated under reducedpressure. The residue was washed with petroleum ether to afford 136c(520 mg, 93%) as a white solid, which was used directed without furtherpurification. MS-ESI: [M+H]⁺ 373.1

Example 136d(S)-2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-(3-methylmorpholine-4-carbonyl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde136d

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 136c (260 mg, 0.70mmol),2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (180 mg, 0.50 mmol), K₃PO₄ (297 mg, 1.4 mmol), sodium acetate (114mg, 1.4 mmol), 1,1′-bis(diphenylphosphino)ferrocenedichloropalladium(II) (57 mg, 0.07 mmol), andacetonitrile/water (10/0.2 mL). After three cycles of vacuum/N₂ flush,the mixture was heated at 100° C. for 1.5 h. Analysis of the reactionmixture by LCMS showed complete conversion to the desired product. Thereaction mixture was cooled to room temperature and concentrated underreduced pressure. The residue was diluted with dichloromethane (20 mL)and water (10 mL). The aqueous layer was separated and extracted withdichloromethane (3×20 mL). The combined organic layers was dried overNa₂SO₄, filtered, and concentrated under reduced pressure. The darkresidue was purified by silica-gel column chromatography eluting with60:1 dichloromethane/methanol to afford 136d (140 mg, 43%) as a yellowsolid. MS-ESI: [M+H]⁺ 652.3

Example 1366-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[[5-[(3S)-3-methylmorpholine-4-carbonyl]-2-pyridyl]amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one136

A solution of 136d (140 mg, 0.21 mmol) in methanol/dichloromethane (4/4mL) was added NaBH₄ (16 mg, 0.63 mmol). The mixture was stirred at roomtemperature for 1 h and quenched with water (2 mL). It was thenevaporated under reduced pressure and the residue was purified byreverse-phase prep-HPLC to afford 136 (40 mg, 28%) as a white solid.MS-ESI: [M+H]⁺ 654.3. ¹H NMR (500 MHz, DMSO-d₆) δ 9.00 (s, 1H), 8.77 (d,J=8.0 Hz, 1H), 8.57 (d, J=5.0 Hz, 1H), 8.53 (d, J=2.5 Hz, 1H), 8.25 (d,J=2.5 Hz, 1H), 7.90 (d, J=1.5 Hz, 1H), 7.78 (d, J=1.0 Hz, 1H), 7.65-7.63(m, 1H), 7.59 (d, J=2.5 Hz, 1H), 7.53 (d, J=5.0 Hz, 1H), 7.37 (d, J=8.5Hz, 1H), 4.91-4.89 (m, 1H), 4.45-4.39 (m, 2H), 4.17-4.13 (m, 1H),3.79-3.67 (m, 2H), 3.58-3.53 (m, overlap, 5H), 3.41-3.36 (m, 1H),3.29-3.25 (m, 1H), 1.39 (s, 9H), 1.22 (d, J=7.5 Hz, 3H).

Example 138a (S)-tert-Butyl4-(6-(5-Chloro-2-methoxypyridin-3-ylamino)pyridin-3-yl)-3-methylpiperazine-1-carboxylate138a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (40 mL),(S)-tert-butyl 4-(6-amino pyridin-3-yl)-3-methylpiperazine-1-carboxylate105b (2.04 g, 7.0 mmol), 3-bromo-5-chloro-2-methoxypyridine (2.8 g, 12.6mmol), Pd₂(dba)₃ (640 mg, 0.70 mmol), XantPhos (404.6 mg, 0.70 mmol),and cesium carbonate (4.56 g, 14.0 mmol). After three cycles ofvacuum/argon flush, the mixture was heated at 100° C. for 4 h. Afterthis time the reaction was cooled to room temperature. It was thenfiltered and the filtrate was evaporated under reduced pressure. Theresidue was purified by silica-gel column chromatography eluting with1:3 ethyl acetate/petroleum ether to afford 138a (1.7 g, 57%) as ayellow solid. MS-ESI: [M+H]⁺ 434.2

Example 138b(S)-5-Chloro-3-(5-(2-methylpiperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one138b

A solution of 138a (1.0 g, 2.3 mmol) in dioxane/HCl (30 mL) was stirredat 100° C. for 2 h. It was then evaporated under reduced pressure toafford 138b (1.48 g, crude) as a yellow solid. MS-ESI: [M+H]⁺ 320.3

Example 138c(S)-5-Chloro-3-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one138c

To a solution of 138b (1.0 g, 3.1 mmol) in methanol (35 mL) was addedoxetan-3-one (669.6 mg, 9.3 mmol), NaBH₃CN (585.9 mg, 9.3 mmol), andZnCl₂ (1.26 g, 9.3 mmol). The reaction was stirred at 30° C. for 2 h.The mixture was evaporated under reduced pressure and the residuediluted with water (20 mL). It was then extracted with dichloromethane(3×30 mL). The combined dichloromethane extract was concentrated underreduced pressure and the residue was purified by silica-gel columnchromatography eluting with 40:1 ethyl acetate/methanol to afford 138c(291 mg, 25%) as a red solid. MS-ESI: [M+H]⁺ 376.3

Example 138(S)-6-tert-Butyl-8-fluoro-2-(3-(hydroxymethyl)-4-(5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-2-yl)phthalazin-1(2H)-one 138

A sealed tube equipped with a magnetic stirrer was charged with 138c(150 mg, 0.40 mmol),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicacid 116c (495.6 mg, 1.2 mmol), Pd₂(dba)₃ (36.6 mg, 0.040 mmol),P(cy)₃(44.6 mg, 0.16 mmol), Cs₂CO₃ (391.2 mg, 1.2 mmol), dioxane (8 mL),and water (0.2 mL). After three cycles of vacuum/argon flush, themixture was heated at 120° C. for 4 h. After this time the reaction wascooled to room temperature. It was then filtered and the filtrate wasevaporated under reduced pressure. The residue was purified bysilica-gel column chromatography eluting with 40:1 ethylacetate/methanol and further purified by reverse-phase prep-HPLC toafford 138 (48 mg, 18%) as a yellow solid. MS-ESI: [M+H]⁺ 667.3. ¹H NMR(500 MHz, DMSO-d₆) δ 12.05 (s, 1H), 8.65 (d, J=2.0 Hz, 1H), 8.57 (d,J=5.0 Hz, 1H), 8.55 (d, J=3.0 Hz, 1H), 8.44 (s, 1H), 7.91 (d, J=2.0 Hz,1H), 7.87 (d, J=2.5 Hz, 1H), 7.79 (dd, J=1.0 Hz, 13.0 Hz, 1H), 7.54 (d,J=5.0 Hz, 1H), 7.39 (dd, J=2.5 Hz, 9.0 Hz, 1H), 7.26-7.23 (m, 2H),4.97-4.95 (m, 1H), 4.58-4.54 (m, 2H), 4.48-4.46 (m, 1H), 4.43-4.41 (m,1H), 4.38-4.37 (m, 2H), 3.69-3.68 (m, 1H), 3.41-3.39 (m, 1H), 3.11-3.09(m, 1H), 2.97-2.93 (m, 1H), 2.56-2.54 (m, 1H), 2.35-2.32 (m, 2H),2.21-2.17 (m, 1H), 1.40 (s, 9H), 0.94 (d, J=6.5 Hz, 3H),

Example 139a (3-Nitro-1H-pyrazol-5-yl)methanol 139a

A mixture of 3-nitro-1H-pyrazole-5-carboxylic acid (4.71 g, 30 mmol),BH₃/THF (75 mL, 1 mol/L, 75 mmol) was stirred at 60° C. for 2 h. Themixture was cooled to room temperature and 4M HCl (19 mL, 75 mmol) wasadded. It was stirred at 70° C. for 2 h. After cooling down to roomtemperature, the mixture was concentrated under reduced pressure. Theresidue was partitioned between ethyl acetate and brine (100:100 mL).The aqueous phase was extract with ethyl acetate (4×50 mL). The combinedorganic layer was dried on Na₂SO₄ and evaporated under reduced pressure.The residue was purified by silica-gel column chromatography elutingwith petroleum ether/ethyl acetate (5:1 to 1:1) to afford 139a (3.5 g,79%) as a white solid. MS-ESI: [M+H]⁺ 144.2

Example 139b1-(5-(Hydroxymethyl)-3-nitro-1H-pyrazol-1-yl)-2-methylpropan-2-ol 139b

A sealed tube was charged with 139a (2.145 g, 15 mmol), Cs₂CO₃ (978 mg,3.0 mmol), and 2,2-dimethyloxirane (15 mL). The mixture was stirred at70° C. for 3 h. After cooled to room temperature, the mixture wasconcentrated under reduced pressure. The residue was purified bysilica-gel column chromatography eluting with petroleum ether/ethylacetate (5:1 to 1:1) to afford 139b (1.2 g, 38%) as a white solid.MS-ESI: [M+H]⁺ 216.2

Example 139c6,6-Dimethyl-2-nitro-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazine 139c

To a solution of 139b (1.1 g, 5.1 mmol) in DMF (10 mL), was added NaH(60 percent dispersion in mineral oil, 246 mg, 6.14 mmol) at 0° C. Theresulting suspension was stirred for 30 min, followed by the addition ofp-toluenesulfonyl chloride (1169 mg, 6.14 mmol). The mixture was stirredat 60° C. overnight. After cooling to room temperature, saturatedammonium chloride solution was added and the mixture was extracted withdichloromethane. The combined organic layer was washed with brine, driedover Na₂SO₄, and evaporated under reduced pressure. The residue waspurified by silica-gel column chromatography eluting with petroleumether/ethyl acetate gradient (9:1 to 2:1) to afford 139c (228 mg, 22%).MS-ESI: [M+H]⁺ 198.3

Example 139d6,6-Dimethyl-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-amine 139d

A 50-mL single-neck round-bottomed flask was purged with nitrogen andcharged with 139c (0.21 g, 1.25 mmol), 10% palladium on carbon (50% wet,125 mg), and methanol (10 mL). The mixture was evacuated, charged withhydrogen gas, and stirred at room temperature for 2 h. The hydrogen wasthen evacuated and nitrogen was charged into the flask. The catalyst wasremoved by filtration through a pad of CELITE® and the filtrate wasconcentrated under reduced pressure to afford 139d (167 mg, 93%).MS-ESI: [M+H]⁺ 168.1

Example 139e5-Bromo-3-(6,6-dimethyl-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methylpyridin-2(1H)-one139e

A 100-mL round-bottomed flask equipped with a reflux condenser wascharged with 1,4-dioxane (10 mL), 139d (167 mg, 1.0 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (320 mg, 1.2 mmol), Pd₂(dba)₃ (91mg, 0.10 mmol), XantPhos (116 mg, 0.20 mmol), and cesium carbonate (652mg, 2.0 mmol). After three cycles of vacuum/argon flush, the mixture washeated at 100° C. for 3 h. It was then filtered and the filtrate wasevaporated under reduced pressure. The residue was purified bysilica-gel column chromatography eluting with 100:1dichloromethane/methanol to afford 139e (210 mg, 60%) as a yellow solid.MS-ESI: [M+H]⁺ 352.9

Example 139f(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(6,6-dimethyl-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 139f

A 25-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 139e (177 mg, 0.50mmol),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicacid 116c (207 mg, 0.50 mmol), Pd(dppf)Cl₂ (41 mg, 0.050 mmol), sodiumacetate (82 mg, 1.0 mmol), K₃PO₄.trihydrate (266 mg, 1.0 mmol), water (6drops), and acetonitrile (5 mL). After three cycles of vacuum/argonflush, the mixture was heated at 100° C. for 2 h. It was then filteredand the filtrate was evaporated under reduced pressure. The residue waspurified by silica-gel column chromatography eluting with 50:1dichloromethane/methanol to afford 139f (161 mg, 50%) as a brown solid.MS-ESI: [M+H]⁺ 642.3

Example 1396-tert-butyl-2-[4-[5-[(6,6-dimethyl-4,7-dihydropyrazolo[5,1-c][1,4]oxazin-2-yl)amino]-1-methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one139

A mixture of 139f (160 mg, 0.25 mmol) and lithium hydroxide (60 mg, 2.5mmol) in i-propanol/THF (1:1, 4 mL) and water (1 mL) was stirred at 30°C. for 1 h. The mixture was evaporated under reduced pressure and theresidue was diluted with water (5 mL). It was then extracted with ethylacetate (3×10 mL). The combined ethyl acetate extract was concentratedunder reduced pressure and the residue was purified by reverse-phaseprep-HPLC to afford 139 (60 mg, 40%) as a light yellow solid. MS-ESI:[M+H]⁺ 599.8. ¹H NMR (500 MHz, DMSO-d₆) δ 8.57 (d, J=5.0 Hz, 1H), 8.54(d, J=2.5 Hz, 1H), 8.34 (s, 1H), 8.08 (d, J=2.5 Hz, 1H), 7.90 (d, J=1.5Hz, 1H), 7.79-7.76 (m, 1H), 7.51 (d, J=5.0 Hz, 1H), 7.39 (d, J=2.0 Hz,1H), 5.95 (s, 1H), 4.92 (bs, 1H), 4.73 (s, 2H), 4.42 (s, 2H), 3.80 (s,2H), 3.60 (s, 3H), 1.40 (s, 9H), 1.26 (s, 6H).

Example 140a5-Bromo-1-methyl-3-(1-methyl-1H-pyrazol-3-ylamino)pyridin-2(1H)-one 140a

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (100 mL),1-methyl-1H-pyrazol-3-amine (970 mg, 10.0 mmol),3,5-dibromo-1-methylpyridin-2-(1H)-one (2.9 g, 11 mmol), and cesiumcarbonate (6.5 g, 20.0 mmol). After bubbling nitrogen through thesuspension for 10 minutes, tris(dibenzylideneacetone)dipalladium(0) (457mg, 0.50 mmol) and Xantphos (587 mg, 1.0 mmol) were added. The systemwas subjected to three cycles of vacuum/argon flush and heated at refluxfor 2 h. It was then cooled to room temperature and filtered. The solidwas washed with dichloromethane (2×50 mL) and the combined organicfiltrate was concentrated. The residue was purified by silica-gel columnchromatography eluting with 30:1 dichloromethane/methanol to afford 140aas a yellow solid (900 mg, 32%). MS-ESI: [M+H]⁺ 283.1

Example 140b1-Methyl-3-(1-methyl-1H-pyrazol-3-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one140b

A 100-mL round-bottomed flask equipped with a reflux condenser wascharged with 140a (564 mg, 2.0 mmol), bis(pinacolato) diboron (1.5 g,6.0 mmol), Pd₂(dba)₃ (183 mg, 0.20 mmol), X-phos (95 mg, 0.20 mmol),potassium acetate (392 mg, 4.0 mmol), and 1,4-dioxane (20 mL). Afterthree cycles of vacuum and argon flush, the mixture was heated at 70° C.for 2 h. Then it was cooled to room temperature and filtered. Thefiltrate was evaporated under reduced pressure. The residue was washedwith petroleum ether to afford 140b (600 mg, 91%) as a brown solid,which was used without further purification. MS-ESI: [M+H]⁺ 331.0

Example 140c2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(1-methyl-1H-pyrazol-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde140c

A 50-mL round-bottomed flask equipped with a reflux condenser wascharged with2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (359 mg, 1.0 mmol), 140b (660 mg, 2.0 mmol), K₃PO₄ (424 mg, 2.0mmol), 1,1′-bis(diphenylphosphino)ferrocenedichloropalladium(II) (82 mg,0.10 mmol), sodium acetate (164 mg, 2.0 mmol), and acetonitrile/water(15/0.2 mL). After three cycles of vacuum/N₂ flush, the mixture washeated at 100° C. for 1.5 h. The mixture was cooled to room temperatureand concentrated under reduced pressure. The residue was diluted withdichloromethane (30 mL) and water (30 mL). The organic layer wasseparated and the aqueous extracted with dichloromethane (3×20 mL). Thecombined organic layer was dried over Na₂SO₄, filtered, and concentratedunder reduced pressure. The dark residue was purified by silica-gelcolumn chromatography eluting with dichloromethane/methanol (80/1 to50/1) to afford 140c (280 mg, 53%) as yellow solid. MS-ESI: [M+H]⁺ 528.1

Example 1406-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[(1-methylpyrazol-3-yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one140

To a solution of 140c (270 mg, 0.50 mmol) in THF (5 mL), propan-2-ol (5mL), and water (2 mL) was added lithium hydroxide (36 mg, 1.5 mmol). Thereaction mixture was stirred at room temperature for 1 h andconcentrated under reduced pressure. The residue was diluted withdichloromethane (20 mL) and water (10 mL). The organic layer wasseparated and the aqueous layer was extracted with dichloromethane (3×15mL). The combined organic layer was washed with brine and concentratedunder reduced pressure. The residue was purified by reverse-phaseprep-HPLC to afford 140 (53 mg, 20%) as a white solid. MS-ESI: [M+H]⁺530.3. ¹H NMR (500 MHz, DMSO-d₆) δ 8.56 (d, J=5.0 Hz, 1H), 8.53 (d,J=2.0 Hz, 1H), 8.21 (s, 1H), 8.03 (d, J=2.5 Hz, 1H), 7.89 (s, 1H),7.78-7.5 (m, 1H), 7.51-7.48 (m, 2H), 7.39 (d, J=2.5 Hz, 1H), 6.07 (d,J=2.0 Hz, 1H), 4.91 (s, 1H), 4.42 (s, 2H), 3.71 (s, 3H), 3.58 (s, 3H),1.39 (s, 9H)

Example 141a6-Chloro-2-methyl-4-(5-methylisoxazol-3-ylamino)pyridazin-3(2H)-one 141a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (40 mL),5-methylisoxazol-3-amine (559 mg, 5.7 mmol),4-bromo-6-chloro-2-methylpyridazin-3(2H)-one (1.89 g, 8.55 mmol),Pd₂(dba)₃ (521 mg, 0.57 mmol), XantPhos (329 mg, 0.57 mmol), and cesiumcarbonate (3.72 g, 11.4 mmol). After three cycles of vacuum/argon flush,the mixture was heated at 100° C. for 3 h. Then the mixture was cooledto 80° C. and filtered. The filtrate was cooled to room temperature. Itwas then filtered to afford 141a (600 mg, 44%) as a yellow solid.MS-ESI: [M+H]⁺ 241.0

Example 141b(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-methylisoxazol-3-ylamino)-6-oxo-1,6-dihydropyridazin-3-yl)pyridin-3-yl)methylAcetate 141b

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 141a (288 mg, 1.2 mmol),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxo-phthalazin-2(1H)-yl)pyridin-4-ylboronicacid 116c (1.49 g, 3.6 mmol), Pd(dppf)Cl₂ (99 mg, 0.12 mmol), potassiumacetate (235 mg, 2.4 mmol), K₃PO₄ (523 mg, 2.4 mmol), acetonitrile (20mL), and water (10 drops). After three cycles of vacuum/argon flush, themixture was heated at 95° C. for 2 h. It was then filtered and thefiltrate was concentrated under reduced pressure. The residue waspurified by silica-gel column chromatography eluting with 1:1 ethylacetate/petroleum ether to afford 141b (220 mg, 32%) as a yellow solid.MS-ESI: [M+H]⁺ 574.2

Example 1416-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[(5-methylisoxazol-3-yl)amino]-6-oxo-pyridazin-3-yl]-2-pyridyl]phthalazin-1-one141

A mixture of 141a (117.3 mg, 0.20 mmol) and lithium hydroxidemonohydrate (84 mg, 2.0 mmol) in i-propanol/THF (1:1, 8 mL) and water (1mL) was stirred at 35° C. for 0.5 h. The mixture was concentrated underreduced pressure. The residue was partitioned between water (10 mL) anddichloromethane (10 mL). The aqueous phase was extracted withdichloromethane (3×10 mL). The combined organic layer was concentratedunder reduced pressure and the residue was purified by reverse-phaseprep-HPLC to afford 141 (70 mg, 66%) as a pale yellow solid. MS-ESI:[M+H]⁺ 532.2. ¹H NMR (500 MHz, DMSO-d₆) δ 9.92 (s, 1H), 8.65 (d, J=5.5Hz, 1H), 8.55 (d, J=2.5 Hz, 1H), 7.90 (d, J=1.5 Hz, 1H), 7.85 (s, 1H),7.80-7.77 (m, 1H), 7.62 (d, J=5.0 Hz, 1H), 6.33 (s, 1H), 4.85 (s, 1H),4.56-4.53 (m, 1H), 4.45-4.42 (m, 1H), 3.80 (s, 3H), 2.36 (s, 3H), 1.40(s, 9H).

Example 142a 3-Bromo-5-iodopyridin-2-ol 142a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with acetonitrile (50 mL), TFA (10 mL),3-bromopyridin-2-ol (4.0 g, 11.56 mmol), N-iodosuccinimide (5.2 g, 11.56mmol). The mixture was stirred at room temperature for 15 h. The mixturewas diluted with water (100 mL) and resulting white solid was collectedby filtration to afford 142a (6.6 g, 96%) as a white solid. MS-ESI:[M+H]⁺ 300

Example 142b 3-Bromo-5-iodo-1-methylpyridin-2(1H)-one 142b

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with DMF (50 mL), 142a (6.0 g, 20.0 mmol), CH₃I(4.26 g, 30.0 mmol), and K₂CO₃ (5.52 g, 40.0 mmol). The mixture wasstirred at room temperature for 2 h and diluted with water (200 mL). Theresulting white solid was collected by filtration to afford 142b (5.97g, 95%) as a white solid. MS-ESI: [M+H]⁺ 314

Example 142c(4-(5-Bromo-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-3-yl)methylacetate 142c

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 142b, (1.37 g, 4.38mmol, 1.0 eq.),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridine-4-ylboronicacid 116c (2.07 mg, 5.0 mmol), Pd(dppf)Cl₂ (179 mg, 0.219 mmol, 0.050eq.), sodium acetate (718 mg, 8.76 mmol, 2.0 eq.), K₃PO₄ (1.86 g, 8.76mmol, 2.0 eq.), acetonitrile (20 mL), and water (1 mL). After threecycles of vacuum/argon flush, the mixture was heated at 30° C. for 2 h.It was then cooled to room temperature and filtered. The filtrate wasconcentrated under reduced pressure and the resulting residue waspurified by silica-gel column chromatography eluting with 100:1dichloromethane/ethanol to afford 142c (800 mg, 32%) as a yellow solid.MS-ESI: [M+H]⁺ 555.2. ¹H NMR (500 MHz, DMSO-d₆) δ 8.66 (d, J=5.0 Hz,1H), 8.56 (d, J=2.5 Hz, 1H), 8.14 (d, J=2.5 Hz, 1H), 8.08 (d, J=2.0 Hz,1H), 7.91 (d, J=1.5 Hz, 1H), 7.82-7.79 (m, 1H), 7.62 (d, J=6.5 Hz, 1H),5.02 (s, 2H), 3.59 (s, 3H), 1.78 (s, 3H), 1.40 (s, 9H).

Example 142d(2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(1-ethyl-1H-pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylacetate 142d

Into a 1-dram vial was added 142c (40 mg, 0.074 mmol),1-ethyl-1H-pyrazol-3-amine (1.2 equiv), cesium carbonate (1.5 equiv),Xantphos (10 mol %) and tris(dibenzylideneacetone) dipalladium(0) (5 mol%) in dry 1,4-dioxane (0.2 M). The reaction was then stirred at 80° C.for 3 hours. After cooling to room temperature, the reaction was thendiluted with dichloromethane (3 mL) and washed with water (2×3 mL). Theorganic layer was dried over magnesium sulfate, filtered andconcentrated in vacuo. The crude product 142d was then carried on to thesubsequent step without purification.

Example 1426-tert-butyl-2-[4-[5-[(1-ethylpyrazol-3-yl)amino]-1-methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one142

Into a 1 dram vial was added 142d (1 equiv) in a 4:1 mixture of THF andwater (1 mL) Lithium hydroxide (1.5 equiv) was then added to the mixtureand the reaction was stirred at room temperature for 16 hours. Thereaction was then diluted with dichloromethane (3 mL) and washed withwater (2×3 mL). The organic layer was collected, dried over magnesiumsulfate, filtered, and concentrated in vacuo. The crude material waspurified by reverse-phase chromatography to give 142 (7.6 mg, 19%yield). ¹H NMR (400 MHz, DMSO-d6) δ 8.55 (s, 1H), 8.52 (d, J=2.5 Hz,1H), 8.16 (s, 1H), 8.06 (d, J=2.5 Hz, 1H), 7.89 (m, 1H), 7.75 (dd,J=13.1, 1.9 Hz, 1H), 7.53 (d, J=2.2 Hz, 1H), 7.51 (d, J=12.6 Hz, 1H),7.40 (d, J=2.1 Hz, 1H), 6.07 (s, 1H), 4.46 (br s 1H), 4.00 (q, J=7.2 Hz,2H), 3.60 (s, 3H), 3.17 (s, 2H), 1.39 (s, 9H), 1.34 (t, J=7.2 Hz, 3H).ES-MS m/z 544.3 [M+1].

Example 1436-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[5-[[5-(methoxymethyl)-1-methyl-pyrazol-3-yl]amino]-1-methyl-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one143

Following the procedures of Example 142, and substituting5-(methoxymethyl)-1-methyl-1H-pyrazol-3-amine for1-ethyl-1H-pyrazol-3-amine, 143 was prepared (11.2 mg, 54% yield). ¹HNMR (400 MHz, DMSO-d6) δ 8.56 (d, J=5.0 Hz, 1H), 8.53 (d, J=2.5 Hz, 1H),8.19 (s, 1H), 8.03 (d, J=2.4 Hz, 1H), 7.89 (d, J=1.8 Hz, 1H), 7.76 (dd,J=13.2, 1.7 Hz, 1H), 7.50 (d, J=5.0 Hz, 1H), 7.39 (d, J=2.4 Hz, 1H),6.11 (s, 1H), 4.43 (d, J=5.4 Hz, 2H), 4.38 (s, 2H), 3.66 (s, 3H), 3.59(s, 3H), 3.26 (s, 3H), 1.39 (s, 9H). ES-MS m/z 574.3 [M+1].

Example 1446-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[[1-methyl-5-(pyrrolidine-1-carbonyl)pyrazol-3-yl]amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one144

Following the procedures of Example 142, and substituting(3-amino-1-methyl-1H-pyrazol-5-yl)(pyrrolidin-1-yl)methanone for1-ethyl-1H-pyrazol-3-amine, 144 was prepared (14.6 mg, 65% yield). ¹HNMR (400 MHz, DMSO-d₆) δ 8.59-8.50 (m, 2H), 8.28 (s, 1H), 8.04 (d, J=2.3Hz, 1H), 7.89 (d, J=1.8 Hz, 1H), 7.76 (dd, J=13.1, 1.7 Hz, 1H), 7.51 (d,J=5.1 Hz, 1H), 7.42 (d, J=2.3 Hz, 1H), 6.46 (s, 1H), 4.87 (t, J=5.1 Hz,1H), 4.47-4.40 (m, 2H), 3.79 (s, 3H), 3.60 (s, 3H), 3.48 (dd, J=19.0,6.7 Hz, 4H), 1.91-1.82 (m, 4H), 1.39 (s, 9H). ES-MS m/z 627.4 [M+1].

Example 1456-tert-butyl-2-[4-[5-[[1-(2,2-difluoroethyl)-5-methyl-pyrazol-3-yl]amino]-1-methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one145

Following the procedures of Example 142, and substituting1-(2,2-difluoroethyl)-5-methyl-1H-pyrazol-3-amine for1-ethyl-1H-pyrazol-3-amine, 145 was prepared (11 mg, 26% yield). ¹H NMR(400 MHz, DMSO-d6) δ 8.55 (d, J=5.0 Hz, 1H), 8.52 (d, J=2.7 Hz, 1H),8.20 (s, 1H), 8.11 (d, J=2.4 Hz, 1H), 7.90 (d, J=1.6 Hz, 1H), 7.75 (dd,J=13.1, 1.8 Hz, 1H), 7.50 (d, J=5.0 Hz, 1H), 7.42 (d, J=2.4 Hz, 1H),5.97 (s, 1H), 4.88 (t, J=5.0 Hz, 1H), 4.46-4.33 (m, 5H), 3.59 (s, 3H),2.21 (s, 3H), 1.39 (s, 9H). ES-MS m/z 594.3 [M+1].

Example 146a Ethyl 2-(5-(Hydroxymethyl)-3-nitro-1H-pyrazol-1-yl)acetate146a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with acetonitrile (30 mL),(3-nitro-1H-pyrazol-5-yl)methanol (1.43 g, 10.0 mmol), Cs₂CO₃ (490 mg,1.5 mmol), and ethyl 2-bromoacetate (2.00 g, 12 mmol). The mixture wasstirred at 40° C. for 5 h. It was then cooled to room temperature andfiltered. The filtrate was concentrated under reduced pressure and theresidue was purified by silica-gel column chromatography eluting with30:1 dichloromethane/methanol to afford 146a (1.65 g, 72%) as a yellowsolid. MS-ESI: [M+H]⁺ 229.9

Example 146b Ethyl 2-(5-(Chloromethyl)-3-nitro-1H-pyrazol-1-yl)acetate146b

To a mixture of 146a (1.50 g, 6.55 mmol) in CHCl₃ (60 mL) cooled at 0°C. was slowly added SOCl₂ (2.34 g, 19.6 mmol) while maintaining theinternal temperature below 5° C. This reaction mixture was warmed to 50°C. and stirred at this temperature for 3 h. It was then cooled to 0° C.and quenched with water. The organic layer was separated and evaporatedunder reduced pressure. The residue was purified by silica-gel columnchromatography eluting with 30:1 dichloromethane/methanol to afford 146b(1.1 g, 68%) as a yellow solid. MS-ESI: [M+H]⁺ 247.9

Example 146c5-Methyl-2-nitro-4,5-dihydropyrazolo[1,5-a]pyrazin-6(7H)-one 146c

To a solution of 146b (1.0 g, 4.0 mmol) in dichloromethane (30 mL) wasadded a solution of CH₃NH₂ (1.07 g, 12.0 mmol, 35% in methanol). Thisreaction mixture was stirred at room temperature for 3 h and dilutedwith water (30 mL). The organic layer was separated, dried over Na₂SO₄,and concentrated under reduced pressure. The residual was purified bysilica-gel column chromatography eluting with 30:1dichloromethane/methanol to afford 146c (450 mg, 57%) as a yellow solid.MS-ESI: [M+H]⁺ 196.9

Example 146d2-Amino-5-methyl-4,5-dihydropyrazolo[1,5-a]pyrazin-6(7H)-one 146d

A solution of 146c (450 mg, 2.3 mmol) in ethanol (30 mL) was added Pd/C(10%, 400 mg). The reaction was charged with hydrogen gas (via balloon)and stirred at room temperature for 2 h. After reaction was complete,the mixture was filtered through a plug of CELITE® and the filtrate wasconcentrated under reduced pressure to afford 146d as a yellow solid(320 mg, 84%), which was used without further purification in the nextstep. MS-ESI: [M+H]⁺ 167.1

Example 146e2-(5-Bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)-5-methyl-4,5-dihydropyrazolo[1,5-a]pyrazin-6(7H)-one146e

A 100-mL round-bottomed flask equipped with a magnetic stirrer and areflux condenser was charged with 146d (300 mg, 1.8 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (482 mg, 1.8 mmol), cesiumcarbonate (1.17 g, 3.6 mmol), and 1,4-dioxane (20 mL). After bubblingnitrogen through the suspension for 10 minutes, xantphos (104 mg, 0.18mmol) and tris(dibenzylideneacetone)dipalladium(0) (82 mg, 0.090 mmol)were added. The system was subjected to three cycles of vacuum/argonflush and heated at reflux for 5 h. It was then cooled to roomtemperature and filtered. The solid was washed with dichloromethane(2×30 mL). The combined filtrate was concentrated under reducedpressure. The residue was purified by silica-gel column chromatographyeluting with dichloromethane/methanol (80/1 to 30/1) to afford 146e (390mg, 61%) as a yellow solid.

Example 146f(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-methyl-6-oxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 146f

A 25-mL round-bottomed flask equipped with a reflux condenser wascharged with3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyri-din-4-ylboronicacid 116c (165 mg, 0.40 mmol), 146e (141 mg, 0.40 mmol), K₃PO₄ (170 mg,0.80 mmol), sodium acetate (66 mg, 0.80 mmol), Pd(dppf)Cl₂ (15 mg, 0.020mmol), and acetonitrile/water (8/0.2 mL). The mixture was subjected tothree cycles of vacuum/nitrogen flush and heated at 100° C. under N₂protection for 1 h. Analysis of the reaction mixture by LCMS showedcomplete conversion to the desired product. The reaction mixture wascooled to room temperature and concentrated under reduced pressure. Theresidue was diluted with dichloromethane (30 mL) and water (30 mL). Theorganic layer was separated and the water layer was extracted withdichloromethane (2×30 mL). The combined organic extract was dried overNa₂SO₄, filtered, and concentrated under reduced pressure. The darkresidue was purified by silica-gel column chromatography eluting withdichloromethane/methanol (80/1 to 30/1) to afford 146f (115 mg, 45%) asa yellow solid. MS-ESI: [M+H]⁺ 641.4

Example 1466-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[(5-methyl-6-oxo-4,7-dihydropyrazolo[1,5-a]pyrazin-2-yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one146

To a solution of 146f (110 mg, 0.172 mmol) in THF/i-propanol/water(4/2/2 mL) was added lithium hydroxide (21 mg, 0.86 mmol). The mixturewas stirred at 30° C. for 1 h. After the reaction was complete, themixture was concentrated under reduced pressure. The residue was dilutedwith water (10 mL) and extracted with ethyl acetate (3×20 mL). Thecombined organic layer was dried and concentrated under reducedpressure. The residue was purified by reverse-phase prep-HPLC to afford146 as a white solid (45 mg, 44%). MS-ESI: [M+H]⁺ 599.2. ¹H NMR (500MHz, DMSO-d₆) δ 8.57 (d, J=5.0 Hz, 1H), 8.54 (d, J=2.5 Hz, 1H), 8.41 (s,1H), 8.08 (d, J=2.5 Hz, 1H), 7.91 (d, J=1.5 Hz, 1H), 7.78 (d, J=13.0 Hz,1H), 7.51 (d, J=5.0 Hz, 1H), 7.42 (d, J=2.5 Hz, 1H), 6.08 (s, 1H),4.91-4.89 (m, 1H), 4.62 (s, 2H), 4.57 (s, 2H), 4.45-4.43 (m, 2H), 3.61(s, 3H), 2.98 (s, 3H), 1.40 (s, 9H).

Example 147a 1-(6-Nitropyridin-3-yl)azetidin-3-ol 147a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with acetonitrile (50 mL),5-fluoro-2-nitropyridine (1.2 g, 7.9 mmol), K₂CO₃ (2.1 g, 15.8 mmol),and azetidin-3-ol hydrochloride (1.3 g, 11.9 mmol). The mixture washeated at 60° C. for 1 h. After this time the reaction was cooled toroom temperature. It was then filtered and the filtrate was evaporatedunder reduced pressure. The residue was purified by silica-gel columnchromatography eluting with dichloromethane/methanol (50:1 to 20:1) toafford 147a (1.1 g, 73%) as a yellow solid. MS-ESI: [M+H]⁺ 196.0.

Example 147b 1-(6-Aminopyridin-3-yl)azetidin-3-ol 147b

A 100-mL single-neck round-bottomed flask was purged with nitrogen andcharged with 147a (1.0 g, 5.1 mmol), 10% palladium on carbon (10% wet,100 mg), and ethanol (40 mL). The mixture was evacuated, charged withhydrogen gas, and stirred at room temperature for 5 h. The hydrogen wasthen evacuated and nitrogen was charged into the flask. The catalyst wasremoved by filtration through a pad of CELITE® and the filtrate wasconcentrated under reduced pressure to afford 147b as a yellow solid(792 mg, 85%). MS-ESI: [M+H]+ 166.1.

Example 147c5-Bromo-3-(5-(3-hydroxyazetidin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one147c

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 147b (792 mg, 4.8 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (1.9 g, 7.2 mmol),tris-(dibenzylideneacetone)dipalladium(0) (440 mg, 0.48 mmol), XantPhos(555 mg, 0.96 mmol), Cs₂CO₃ (3.1 g, 9.6 mmol), and 1,4-dioxane (40 mL).After three cycles of vacuum/argon flush, the mixture was heated at 90°C. for 3.0 hrs. It was then cooled to room temperature and filtered. Thefiltrate was concentrated under reduced pressure and the resultingresidue was purified by silica-gel column chromatography eluting withdichloromethane/methanol (50:1 to 20:1) to afford 147c as a yellow solid(1.5 g, 89%). MS-ESI: [M+H]⁺ 351.1

Example 147d(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(5-(3-hydroxyazetidin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 147d

A 100-mL flask equipped with a reflux condenser was charged with 147c(285 mg, 0.81 mmol),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicacid 116c (669 mg, 1.62 mmol), Pd(dppf)Cl₂ (66.2 mg, 0.081 mmol), K₃PO₄(343.4 mg, 1.62 mmol), sodium acetate (132.8 mg, 1.62 mmol), water (0.5mL), and acetonitrile (15 mL). After three cycles of vacuum/argon flush,the mixture was heated at 100° C. for 3 hrs. It was then cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure and the resulting residue was purified by silica-gel columnchromatography eluting with dichloromethane/methanol (50:1 to 30:1) toafford 147d as a brown solid (140.0 mg, 27%). MS-ESI: [M+H]⁺ 640.3.

Example 1476-tert-butyl-8-fluoro-2-[4-[5-[[5-(3-hydroxyazetidin-1-yl)-2-pyridyl]amino]-1-methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]phthalazin-1-one147

A mixture of 147d (140 mg, 0.22 mmol) and lithium hydroxide (132 mg, 5.5mmol) in i-propanol/THF/water (2:2:1, 10 mL) was stirred at roomtemperature for 1 hour. The mixture was concentrated under reducedpressure and diluted with water (10 mL). The resulting mixture wasextracted with dichloromethane three times. The combined organic layerwas concentrated under reduced pressure and the resulting residue waspurified by reverse-phase prep-HPLC to afford 147 (40 mg, 31%) as ayellow solid. MS-ESI: [M+H]⁺ 598.2. ¹H NMR (500 MHz, DMSO-d₆) δ8.57-8.53 (m, 3H), 8.32 (s, 1H), 7.90 (s, 1H), 7.77 (d, J=13.0 Hz, 1H),7.51-7.49 (m, 2H), 7.43 (s, 1H), 7.19 (d, J=8.5 Hz, 1H), 6.88 (dd,J=3.0, 8.5 Hz, 1H), 5.57-5.56 (m, 1H), 4.89-4.87 (m, 1H), 4.55-4.52 (m,1H), 4.43-4.41 (m, 2H), 4.05-4.02 (m, 2H), 3.60 (s, 3H), 3.45-3.42 (m,2H), 1.39 (s, 9H).

Example 148a2-Methyl-1-(2-nitro-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)propan-1-one148a

To a solution of 2-nitro-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine 124d(160 mg, 0.952 mmol) in dichloromethane (10 mL) was added triethylamine(197 mg, 1.90 mmol). After stirring at room temperature for 5 minutes, asolution of isobutyryl chloride (111.0 mg, 1.047 mmol) indichloromethane (2 mL) was added and the mixture was stirred for 1 h.Analysis of the reaction mixture by LCMS showed complete conversion tothe desired product. It was then concentrated under reduced pressure toafford 148a as a white solid (220 mg, 97%), which was used in the nextstep without further purification. MS-ESI: [M+H]⁺ 239.0

Example 148b1-(2-Amino-6,7-dihydropyrazolo[1,5-a]pyrazin-5(4H)-yl)-2-methylpropan-1-one148b

To a solution of 148a (220 mg, 0.924 mmol) in methanol (20 mL) was addedPd/C (22 mg). The system was evacuated and refilled with H₂. Afterstirring at room temperature for 2 h, the mixture was filtered. Thefiltrate was concentrated under reduced pressure to afford 148b as ayellow solid (190 mg, 98%), which was used in the next step withoutfurther purification. MS-ESI: [M+H]⁺ 209.1

Example 148c5-Bromo-3-(5-isobutyryl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino-1-methylpyridin-2(1H)-one148c

A 100-mL round-bottomed flask equipped with a reflux condenser wascharged with 148b (190 mg, 0.913 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (365.6 mg, 1.37 mmol), Pd₂(dba)₃(41.7 mg, 0.0456 mmol), Xantphos (52.8 mg, 0.0913 mmol), Cs₂CO₃ (595.3mg, 1.826 mmol), and dioxane (20 mL). The system was subjected to threecycles of vacuum/nitrogen flush and heated at 100° C. under N₂protection for 3 h. Analysis of the reaction mixture by LCMS showedcomplete conversion to the desired product. It was cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure. The residue was washed with acetonitrile (2 mL) to afford 148cas a dark solid (240 mg, 67%), which was used in the next step withoutfurther purification. MS-ESI: [M+H]⁺ 394.2

Example 148d3-(5-Isobutyryl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one148d

A 100-mL round-bottomed flask equipped with a reflux condenser wascharged with 148c (220 mg, 0.558 mmol), Pin₂B₂ (212.6 mg, 0.837 mmol),Pd₂(dba)₃ (25.5 mg, 0.0279 mmol), X-Phos (26.6 mg, 0.0558 mmol),potassium acetate (109.4 mg, 1.12 mmol), and dioxane (20 mL). The systemwas subjected to three cycles of vacuum/nitrogen flush and heated at 70°C. under N₂ protection for 2 h. Analysis of the reaction mixture by LCMSshowed complete conversion to the desired product. It was then cooled toroom temperature and filtered. The filtrate was concentrated underreduced pressure. The residue was washed with petroleum ether to afford148d as a dark oil (200 mg, 81%), which was used in the next stepwithout further purification. MS-ESI: [M+H]⁺ 442.4

Example 148e2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(5-isobutyryl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde148e

A 100-mL round-bottomed flask equipped with a reflux condenser wascharged with2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (150 mg, 0.42 mmol), 148d (278 mg, 0.63 mmol), Pd(dppf)Cl₂ (17.1mg, 0.021 mmol), K₃PO₄ (178.1 mg, 0.84 mmol), sodium acetate (68.9 mg,0.84 mmol), acetonitrile (15 mL), and water (5 drops). The system wassubjected to three cycles of vacuum/nitrogen flush and heated at 100° C.under N₂ protection for 1 h. Analysis of the reaction mixture by LCMSshowed complete conversion to the desired product. It was cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure. The residue was washed with acetonitrile (0.5 mL) to afford148e as a white solid (100 mg, 37%), which was used in the next stepwithout further purification. MS-ESI: [M+H]⁺ 638.8

Example 1486-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[[5-(2-methylpropanoyl)-6,7-dihydro-4H-pyrazolo[1,5-a]pyrazin-2-yl]amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one148

To a solution of 148e (90 mg, 0.141 mmol) in dichloromethane (5 mL) andmethanol (5 mL) was added NaBH₄ (10.7 mg, 0.282 mmol). After stirring atroom temperature for 1 h, the reaction mixture was quenched with aqueousNH₄Cl and concentrated under reduced pressure. The residue was extractedwith dichloromethane (3×15 mL). The combined organic layer was washedwith brine, dried over Na₂SO₄, and concentrated under reduced pressure.The residue was purified by reverse-phase prep-HPLC to afford 148 (40mg, 44%) as a white solid. MS-ESI: [M+H]⁺ 640.8. ¹H NMR (500 MHz,DMSO-d₆) δ 8.57 (d, J=5.0 Hz, 1H), 8.54 (d, J=2.5 Hz, 1H), 8.35-8.28 (m,1H), 8.06 (s, 1H), 7.90 (d, J=1.5 Hz, 1H), 7.78 (dd, J=1.0, 13.0 Hz,1H), 7.51 (d, J=5.0 Hz, 1H), 7.40 (d, J=2.5 Hz, 1H), 6.01 (s, 1H),4.90-4.88 (m, 1H), 4.78-4.76 (m, 1H), 4.64-4.62 (m, 1H), 4.42-4.41 (m,2H), 4.03-3.92 (m, 4H), 3.59 (s, 3H), 3.00-2.96 (m, 1H), 1.40 (s, 9H),1.04-1.01 (m, 6H).

Example 149a 1-Methyl-4-nitro-1H-1,2,3-triazole 149a

To a 100-mL single-neck round-bottomed containing4-nitro-2H-1,2,3-triazole (2.0 g, 17.5 mmol) and THF (10 mL) at 0° C.was added NaH (1.7 g, 35.0 mmol, 2.0 eq.). The mixture was stirred at 0°C. for 15 min. A solution of CH₃I (3.68 g, 26.3 mmol, 1.5 eq.) inacetone (40 mL) was added and the resulting reaction mixture was stirredat room temperature for 2 h. After this time, the reaction was quenchedby water (20 mL) at 0° C. and concentrated under reduced pressure. Theresidue was diluted with dichloromethane (100 mL). It was then washedwith brine, dried over anhydrous Na₂SO₄, and concentrated under reducedpressure. The residue was purified by silica-gel column chromatographyeluting with 6:1 petroleum ether/ethyl acetate to afford1-methyl-5-nitro-1H-1,2,3-triazole (1.34 g, 60%) as a white solid and149a (800 mg, 35%) as a slightly yellow solid. ¹H NMR (500 MHz, CDCl₃) δ8.34 (s, 1H), 4.26 (s, 3H). 1-Methyl-5-nitro-1H-1,2,3-triazole: ¹H NMR(500 MHz, CDCl₃) δ 8.16 (s, 1H), 4.31 (s, 3H).

Example 149b 1-Methyl-1H-1,2,3-triazol-4-amine 149b

Following the procedure in Example 148b, and starting with 149a (800 mg,6.25 mmol) and 10% palladium on carbon (50% wet, 160 mg), afforded 149bas a yellow solid (600 mg, 98%). ¹H NMR (500 MHz, CDCl₃) δ 6.91 (s, 1H),3.97 (s, 3H), 3.65 (brs, 2H).

Example 149c5-Bromo-1-methyl-3-(1-methyl-1H-1,2,3-triazol-4-ylamino)pyridin-2(1H)-one149c

Following the procedure in Example 148c, and starting with 149b (500 mg,5.10 mmol, 1.0 eq.) and 3,5-dibromo-1-methylpyridin-2(1H)-one (2.04 g,7.65 mmol, 1.5 eq.), 149c was obtained as a yellow solid (760 mg, 52%).MS-ESI: [M+H]⁺ 283.9.

Example 149d(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(1-methyl-1H-1,2,3-triazol-4-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 149d

Following the procedure in Example 147d, and starting with 149c (120 mg,0.42 mmol, 1 eq.) and3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicacid (116c) (485 mg, 1.18 mmol, 2.8 eq.), afforded 149d as a yellowsolid (120 mg, 50%). MS-ESI: [M+H]⁺ 573.3.

Example 1496-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[(1-methyltriazol-4-yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one149

Following the procedure in Example 147, and starting with 149c (90 mg,0.16 mmol), afforded 149 as a white solid (32 mg, 38%). MS-ESI: [M+H]⁺530.8. ¹H NMR (500 MHz, CDCl₃) δ 8.65 (d, J=5.0 Hz, 1H), 8.37 (d, J=1.5Hz, 1H), 7.69-7.67 (m, 3H), 7.60-7.57 (m, 2H), 7.50 (d, J=5.0 Hz, 1H),7.34 (d, J=1.5 Hz, 1H), 4.53 (s, 2H), 4.27 (s, 1H), 4.10 (s, 3H), 3.74(s, 3H), 1.46 (s, 9H).

Example 150a N-Methoxy-N-methyl-3-nitro-1H-pyrazole-5-carboxamide 150a

A 500-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with 3-nitro-1H-pyrazole-5-carboxylic acid (15.7 g,1.0 eq., 100 mmol), N,O-dimethylhydroxylamine hydrochloride (19.5 g, 2.0eq., 200 mmol), HATU (76.0 g, 2.0 eq., 200 mmol), triethylamine (40.4 g,4.0 eq., 400 mmol), and dichloromethane (300 mL). The reaction mixturewas stirred at room temperature overnight. The solvent was removed underreduced pressure. The resulting residue was purified by silica-gelcolumn chromatography eluting with 100:1 dichloromethane/methanol toafford 150a (16.0 g, 80%) as a white solid. MS-ESI: [M+H]⁺ 201.1

Example 150b 3-Amino-N-methoxy-N-methyl-1H-pyrazole-5-carboxamide 150b

A 250-mL single-neck round-bottomed flask was purged with nitrogen andcharged with 150a (16.0 g, 1.0 eq., 80.0 mmol), 10% palladium on carbon(50% wet, 800 mg), and methanol (100 mL). The mixture was evacuated,charged with hydrogen gas, and stirred under hydrogen atmosphere at roomtemperature overnight. The hydrogen was then evacuated and nitrogen wascharged into the flask. The catalyst was removed by filtration through apad of CELITE®. The filtrate was concentrated under reduced pressure toafford 150b (11.0 g, 81%) as a white solid. MS-ESI: [M+H]⁺ 171.1

Example 150c3-(2,5-Dimethyl-1H-pyrrol-1-yl)-N-methoxy-N-methyl-1H-pyrazole-5-carboxamide150c

A 250-mL round-bottomed flask equipped with a magnetic stirrer and aDean-Stark trap was charged with 150b (11.0 g, 1.0 eq., 64.7 mmol),hexane-2,5-dione (11.1 g, 1.5 eq., 97.2 mmol), p-toluenesulfonic acidmonohydrate (558 mg, 0.05 eq., 3.24 mmol), and toluene (100 mL). Thereaction mixture was refluxed overnight. The resulting mixture wascooled to room temperature and concentrated under reduced pressure. Theresulting residue was purified by silica-gel column chromatographyeluting with 1:2 petroleum ether/ethyl acetate to afford 150c (10.4 g,65%) as a white solid. MS-ESI: [M+H]⁺ 249.0

Example 150d 1-(3-(2,5-Dimethyl-1H-pyrrol-1-yl)-1H-pyrazol-5-yl)ethanone150d

A 250-mL round-bottomed flask equipped with a magnetic stirrer wascharged with 150c (7.44 g, 1.0 eq., 30.0 mmol) and THF (100 mL). UnderN₂ protection, a solution of MeMgBr (3.0 Min ether) (25 mL, 2.5 eq.,75.0 mmol) was added at 0° C. The mixture was stirred at roomtemperature for 5 h and quenched with saturated NH₄Cl solution (30 mL).The mixture was concentrated under reduced pressure and the residue wasextracted with ethyl acetate (3×50 mL). The combined organic layer wasevaporated under reduced pressure and the residue was purified bysilica-gel column chromatography eluting with 4:1 petroleum ether/ethylacetate to afford 150d as a white solid (5.40 g, 89%). MS-ESI: [M+H]⁺204.0. ¹H NMR (500 MHz, CDCl₃) δ 6.74 (s, 1H), 5.92 (s, 2H), 2.61 (s,3H), 2.15 (s, 6H).

Example 150e1-(1-(2-Bromoethyl)-3-(2,5-dimethyl-1H-pyrrol-1-yl)-1H-pyrazol-5-yl)ethanone150e

A 250-mL round-bottomed flask equipped with a magnetic stirrer and areflux condenser was charged with 150d (5.4 g, 1.0 eq., 26.6 mmol),1,2-dibromoethane (20.0 g, 4.0 eq., 106.4 mmol), K₂CO₃ (7.34 g, 2.0 eq.,53.2 mmol), and acetonitrile (100 mL). The reaction mixture was refluxfor 5 hrs. It was cooled to room temperature and filtered. The filtratewas concentrated under reduced pressure. The residue was purified bysilica-gel column chromatography eluting with 6:1 petroleum ether/ethylacetate to afford 150e (7.5 g, 91%) as a colorless oil. MS-ESI: [M+H]⁺309.9

Example 150f1-(1-(2-Bromoethyl)-3-(2,5-dimethyl-1H-pyrrol-1-yl)-1H-pyrazol-5-yl)ethanol150f

A 100-mL round-bottomed flask equipped with a magnetic stirrer wascharged with 150e (2.1 g, 1.0 eq., 6.8 mmol), NaBH4 (1.29 g, 5.0 eq.,34.0 mmol), and methanol (50 mL). The mixture was stirred at roomtemperature for 2 h and quenched with water (30 mL). It was thenconcentrated under reduced pressure and the residue was extracted withdichloromethane (3×50 mL). The combined organic layer was concentratedunder reduced pressure to afford crude 150f, which was used in the nextstep without further purification. MS-ESI: [M+H]⁺ 311.9

Example 150g2-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methyl-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazine150g

A 100-mL round-bottomed flask equipped with a reflux condenser wascharged with 150f (2.0 g, 1.0 eq., 6.4 mmol), K₂CO₃ (1.77 g, 2.0 eq.,12.8 mmol), acetonitrile (50 mL). The reaction mixture was refluxovernight. It was then cooled to room temperature and filtered. Thefiltrate was concentrated under reduced pressure. The residue waspurified by silica-gel column chromatography eluting with 4:1 petroleumether/ethyl acetate to afford 150g (550 mg, 37%, over two steps) as acolorless oil. MS-ESI: [M+H]⁺ 232.3

Example 150h 4-Methyl-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-amine150h

A 100-mL round-bottomed flask equipped with a magnetic stirrer and areflux condenser was charged with 150g (550 mg, 1.0 eq., 2.38 mmol),hydroxylamine hydrochloride (827 mg, 5.0 eq., 11.9 mmol), and ethanol(30 mL). The mixture was refluxed for 2 days. It was then cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure. The residue was purified by reverse-phase prep-HPLC to afford150h (30 mg, 8%) as a yellow oil. MS-ESI: [M+H]⁺ 154.1. ¹H NMR (500 MHz,CDCl₃) δ 5.37 (s, 1H), 4.73 (q, J=7.0 Hz, 1H), 4.26-4.23 (m, 1H),4.08-4.03 (m, 1H), 3.91-3.90 (m, 1H), 3.90-3.87 (m, 1H), 1.65 (d, J=7.0Hz, 3H).

Example 150i(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(4-methyl-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylacetate 150i

A 25-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 150h (25 mg, 1.0 eq.,0.16 mmol),(4-(5-bromo-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-3-yl)methylacetate 142c (181 mg, 2.0 eq., 0.32 mmol), Pd₂(dba)₃ (15 mg, 0.1 eq.,0.016 mmol), Xantphos (19 mg, 0.2 eq., 0.032 mmol), Cs₂CO₃ (105 mg, 2.0eq., 0.32 mmol), and dioxane (10 mL). The mixture was subjected to threecycles of vacuum/argon flush and stirred at 100° C. for 2 hr. It wasthen cooled to room temperature and filtered. The filtrate wasconcentrated under reduced pressure. The resulting residue was purifiedby silica-gel column chromatography eluting with 20:1dichloromethane/methanol to afford 150i as a brown solid (48 mg, 48%).

Example 1506-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[(4-methyl-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazin-2-yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one150

A 25-mL round-bottomed flask equipped with a magnetic stirrer wascharged with 150i (48 mg, 1.0 eq., 0.077 mmol), lithium hydroxide (9.2mg, 5.0 eq., 0.38 mmol), i-propanol/THF (4/4 mL), and water (1 mL). Themixture was stirred at room temperature for 1 h and concentrated underreduced pressure. The residue was purified by reverse-phase prep-HPLC toafford 150 as a yellow solid (6.8 mg, 15%). MS-ESI: [M+H]⁺ 585.8. ¹H NMR(500 MHz, CDCl₃) δ 8.66 (d, J=4.5 Hz, 1H), 8.35 (d, J=2.0 Hz, 1H), 8.03(d, J=1.5 Hz, 1H), 7.59-7.53 (m, 4H), 7.47 (s, 1H), 5.72 (s, 1H),4.82-4.78 (m, 1H), 4.52-4.49 (m, 2H), 4.33-4.30 (m, 1H), 4.30-4.27 (m,1H), 4.02-3.98 (m, 3H), 3.73 (s, 3H), 1.54 (d, J=7.0 Hz, 3H), 1.48 (s,9H).

Example 151a Ethyl 2-(5-(Hydroxymethyl)-3-nitro-1H-pyrazol-1-yl)acetate151a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with acetonitrile (30 mL),(3-nitro-1H-pyrazol-5-yl)methanol (1.43 g, 10.0 mmol), Cs₂CO₃ (490 mg,1.5 mmol), and ethyl 2-bromoacetate (2.00 g, 12 mmol). The mixture wasstirred at 40° C. for 5 h. It was then cooled to room temperature andfiltered. The filtrate was concentrated under reduced pressure and theresidue was purified by silica-gel column chromatography eluting with30:1 dichloromethane/methanol to afford 151a (1.65 g, 72%) as a yellowsolid. MS-ESI: [M+H]⁺ 229.9

Example 151b Ethyl 2-(5-(Chloromethyl)-3-nitro-1H-pyrazol-1-yl)acetate151b

To a mixture of 151a (1.50 g, 6.55 mmol) in CHCl₃ (60 mL) cooled at 0°C. was slowly added SOCl₂ (2.34 g, 19.6 mmol) while maintaining theinternal temperature below 5° C. This reaction mixture was warmed to 50°C. and stirred at this temperature for 3 h. It was then cooled to 0° C.and quenched with water. The organic layer was separated and evaporatedunder reduced pressure. The residue was purified by silica-gel columnchromatography eluting with 30:1 dichloromethane/methanol to afford 151b(1.1 g, 68%) as a yellow solid. MS-ESI: [M+H]⁺ 247.9

Example 151c Ethyl2-(5-((Isopropylamino)methyl)-3-nitro-1H-pyrazol-1-yl)acetate 151c

A mixture of 151b (500 mg, 2.04 mmol), propan-2-amine (180 mg, 3.06mmol), and dichloromethane (20 mL) was stirred at room temperature for 2h. The mixture was concentrated under reduced pressure to afford 151c(400 mg, 74%) as a yellow solid, which was used in the next step withoutfurther purification. MS-ESI: [M+H]⁺ 271.1

Example 151d5-Isopropyl-2-nitro-4,5-dihydropyrazolo[1,5-a]pyrazin-6(7H)-one 151d

A mixture of 151c (400 mg, 1.48 mmol) and methanol (30 mL) was stirredat 50° C. for 16h. The mixture was the concentrated under reducedpressure. The residue was diluted with water (80 mL) and extracted withethyl acetate (3×50 mL). The combined organic layer was dried overNa₂SO₄ and concentrated under reduced pressure to afford 151d (300 mg,90%) as a yellow solid. MS-ESI: [M+H]⁺ 225.3

Example 151e2-Amino-5-isopropyl-4,5-dihydropyrazolo[1,5-a]pyrazin-6(7H)-one 151e

To a solution of 151d (220 mg, 1.0 mmol) in ethanol (20 mL) was addedPd/C (10%, 22 mg). The reaction mixture was charged with hydrogen gas(via balloon) and stirred at room temperature for 1 h. The reactionmixture was filtered through a plug of CELITE® and the filtrate wasconcentrated under reduced pressure to afford 151e as a yellow solid(180 mg, 92%), which was used without further purification. MS-ESI:[M+H]⁺ 195.3

Example 151f2-(5-Bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)-5-isopropyl-4,5-dihydropyrazolo[1,5-a]pyrazin-6(7H)-one151f

A 100-mL round-bottomed flask equipped with a reflux condenser wascharged with 151e (200 mg, 1.0 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (268 mg, 1.0 mmol), Pd₂(dba)₃ (50mg, 0.050 mmol), xantphos (58 mg, 0.10 mmol), Cs₂CO₃ (652 mg, 2.0 mmol)and 1,4-dioxane (20 mL). The system was subjected to three cycles ofvacuum/argon flush and heated at 100° C. for 2 h. It was then cooled toroom temperature and filtered. The solid was washed with dichloromethane(2×10 mL). The combined filtrate was concentrated under reducedpressure. The residue was purified by silica-gel column chromatographyeluting with dichloromethane/methanol (80:1 to 30:1) to afford 151f (300mg, 79%) as a yellow solid. MS-ESI: [M+H]⁺ 380.2

Example 151g5-Isopropyl-2-(1-methyl-2-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2-dihydropyridin-3-ylamino)-4,5-dihydropyrazolo[1,5-a]pyrazin-6(7H)-one151g

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 151f (200 mg, 0.52mmol), Pin₂B₂ (330 mg, 1.3 mmol), Pd₂(dba)₃ (25 mg, 0.026 mmol), X-phos(25 mg, 0.052 mmol), potassium acetate (150 mg, 1.5 mmol), and1,4-dioxane (10 mL). The mixture was subjected to three cycles ofvacuum/argon flush and heated at 65° C. for 4 h. It was then filteredand the filtrate was evaporated under reduced pressure. The residue waswashed by petroleum ether to afford crude 151g (300 mg, purity: 50%) asa brown solid. MS-ESI: [M+H]⁺ 428.2.

Example 151h2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(5-isopropyl-6-oxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde151h

A 25-mL round-bottomed flask equipped with a reflux condenser wascharged with2-(6-tert-butyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-4-chloronicotinaldehyde103b (108 mg, 0.30 mmol), 151g (256 mg, 0.60 mmol), K₃PO₄ (127 mg, 0.60mmol), sodium acetate.water (82 mg, 0.60 mmol), Pd(dppf)Cl₂ (12 mg,0.015 mmol), water (0.5 mL), and acetonitrile (8 mL). The reactionmixture was subjected to three cycles of vacuum/nitrogen flush andheated at 80° C. under N₂ protection for 2 h. It was then cooled to roomtemperature and concentrated under reduced pressure. The residue wasdiluted with dichloromethane (20 mL) and water (10 mL). The organiclayer was separated and the water layer was extracted withdichloromethane (2×10 mL). The combined organic extract was dried overNa₂SO₄, filtered, and concentrated under reduced pressure. The darkresidue was purified by silica-gel column chromatography eluting withdichloromethane/methanol (80:1 to 30:1) to afford 151h (90 mg, 48%) asyellow solid. MS-ESI: [M+H]⁺ 625.2.

Example 1516-tert-Butyl-8-fluoro-2-(3-(hydroxymethyl)-4-(5-(5-isopropyl-6-oxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)pyridin-2-yl)phthalazin-1(2H)-one151

A 25-mL single-neck round-bottomed flask was charged with 151h (90 mg,0.14 mmol), NaBH₄ (18 mg, 0.42 mmol), and methanol (5 mL). The mixturewas stirred at room temperature for 1 h and concentrated under reducedpressure. To the resulting residue was added water (10 mL) and themixture was extracted with dichloromethane (3×15 mL). The combinedorganic layer was concentrated under reduced pressure. The residue waspurified by reverse-phase prep-HPLC to afford 151 (50 mg, 55%). MS-ESI:[M+H]⁺ 626.8. ¹H NMR (500 MHz, CDCl₃) δ 8.67 (d, J=5.0 Hz, 1H), 8.36 (d,J=2.5 Hz, 1H), 8.02 (d, J=2.0 Hz, 1H), 7.59-7.58 (m, 3H), 7.55-7.52 (m,2H), 5.90 (s, 1H), 5.07-5.05 (m, 1H), 4.74 (s, 2H), 4.50-4.47 (m, 4H),4.09-4.06 (m, 1H), 3.73 (s, 3H), 1.45 (s, 9H), 1.26 (d, J=7.0 Hz, 6H).

Example 152a tert-Butyl 3-Amino-1H-pyrazole-1-carboxylate 152a

To a mixture of 3-aminopyrazole (3.0 g, 36 mmol) and triethylamine (7.6g, 75 mmol) in 1,4-dioxane (35 mL) was added (Boc)₂O (7.8 g, 36 mmol).The reaction mixture was stirred at 25° C. for 2 h. It was thenconcentrated under reduced pressure. The residue was purified bysilica-gel column eluting with 3:1 petroleum ether/ethyl acetate toafford 152a as a white solid (3.4 g, 52%). MS-ESI: [M+H]⁺ 184.1.

Example 152b 3-(1H-Pyrazol-3-ylamino)-5-bromo-1-methylpyridin-2(1H)-one152a

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 152a (2.2 g, 12 mmol),XantPhos (0.69 g, 1.2 mmol), Pd₂(dba)₃ (1.1 g, 1.2 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (6.4 g, 24 mmol), Cs₂CO₃ (15.6 g,48 mmol), and 1,4-dioxane (50 mL). After bubbling nitrogen through theresulting mixture for 10 minutes, it was heated at 105° C. for 15 h. Themixture was cooled to room temperature and filtered. The filtered wasconcentrated under reduced pressure and the residue the mixture waswashed with methanol (8 mL) to afford 152b as a pale yellow solid (1.2g, 37%). MS-ESI: [M+H]⁺ 269.1.

Example 152c(4-(5-(1H-Pyrazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-3-yl)methylAcetate 152c

A sealed tube equipped with a magnetic stirrer was charged with 152b(200 mg, 0.74 mmol),(2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)methylacetate 116c (370 mg, 0.74 mmol), Pd(dppf)Cl₂ (30 mg, 0.035 mmol),sodium acetate (74 mg, 0.90 mmol), K₃PO₄ (191 mg, 0.90 mmol), andacetonitrile/water (5 mL/0.5 mL). After three cycles of vacuum/argonflush, the mixture was heated at 100° C. for 2 h. It was then filteredand the filtrate was evaporated under reduced pressure. The residue waspurified by silica-gel column chromatography eluting with 10:1dichloromethane/methanol to afford 152c (100 mg, 25%) as a brown solid.MS-ESI: [M+H]⁺ 558.3.

Example 1526-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-6-oxo-5-(1H-pyrazol-3-ylamino)-3-pyridyl]-2-pyridyl]phthalazin-1-one152

A mixture of 152c (100 mg, 0.18 mmol) and lithium hydroxide hydrate (84mg, 2.0 mmol) in THF (8 mL), i-propanol (8 mL) and water (2 mL) wasstirred at 40° C. for 0.5 h. The mixture was evaporated under reducedpressure and the residue was diluted with water (5 mL). It was thenextracted with ethyl acetate (2×10 mL). The combined extract wasconcentrated under reduced pressure and the residue was purified byreverse-phase prep-HPLC to afford 152 (6.5 mg, 7%) as a pale yellowsolid. MS-ESI: [M+H]⁺ 515.8. ¹H NMR (500 MHz, CDCl₃) δ 8.62-8.61 (m,1H), 8.32 (s, 1H), 8.04 (m, 1H), 7.55-7.53 (m, 4H), 7.50-7.49 (m, 1H),7.44 (s, 1H), 6.01 (s, 1H), 4.49-4.48 (m, 2H), 3.71 (s, 3H), 1.45 (s,9H).

Example 153a6-Chloro-2-methyl-4-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridazin-3(2H)-one153a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (30 mL),5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-amine 128b (1.70 g,11.2 mmol), 4-bromo-6-chloro-2-methylpyridazin-3(2H)-one (2.68 g, 12.0mmol), and cesium carbonate (7.30 g, 22.4 mmol). After bubbling nitrogenthrough the suspension for 30 minutes, Xantphos (0.59 g, 1.02 mmol) andtris(dibenzylideneacetone)dipalladium(0) (467 mg, 0.51 mmol) were added.The system was subjected to three cycles of vacuum/argon flush andheated at 90° C. for 2 h. It was then cooled to room temperature andfiltered. The filtrate was evaporated in vacuo. The residue was purifiedby silica-gel column chromatography eluting with 30:1dichloromethane/methanol to afford 153a (1.9 g, 60%) as a brown solid.LCMS: [M+H]⁺ 295.1

Example 153b1-Methyl-5-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1,6-dihydropyridazin-3-ylboronicAcid 153b

A 50-mL round-bottomed flask equipped with a magnetic stirrer and areflux condenser was charged with 153a (200 mg, 0.68 mmol),bis(pinacolato)diboron (Pin₂B₂, 863 mg, 3.40 mmol), Pd₂(dba)₃ (55 mg,0.060 mmol), X-Phos (60 mg, 0.12 mmol), potassium acetate (60 mg, 1.36mmol), and 1,4-dioxane (10 mL). The system was evacuated and thenrefilled with N₂. It was then heated at 50° C. for 2 h. After completionof the reaction, the mixture was filtered and the solid was washed withethyl acetate (10 mL). The combined filtrate was evaporated underreduced pressure to afford 153b as a pale yellow solid, which was usedin the next step. MS-ESI: [M+H]⁺ 305.1.

Example 153c2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1,6-dihydropyridazin-3-yl)nicotinaldehyde153c

A 25-mL round-bottomed flask equipped with a reflux condenser wascharged with 153b (200 mg, 0.66 mmol),2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (240 mg, 0.66 mmol), PdCl₂(dppf) (54 mg, 0.066 mmol), K₃PO₄ (250mg, 1.2 mmol), sodium acetate (100 mg, 1.20 mmol), acetonitrile (10 mL),and water (0.5 mL). The system was evacuated and then refilled with N₂.It was then heated at 100° C. for 1 h. The mixture was cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure and the residue was purified on silica-gel column eluting with20:1 dichloromethane/methanol to afford 153c as a pale yellow solid (170mg, 42%). MS-ESI: [M+H]⁺ 584.3.

Example 1536-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[(5-methyl-6,7-dihydro-4H-pyrazolo[1,5-a]pyrazin-2-yl)amino]-6-oxo-pyridazin-3-yl]-2-pyridyl]phthalazin-1-one153

A mixture of 153c (170 mg, 0.29 mmol) and NaBH₄(20 mg, 0.50 mmol) inmethanol (5 mL) was stirred at room temperature for 0.5 h. The reactionmixture was then quenched with water (7 mL) and concentrated underreduced pressure. The residue was extracted with dichloromethane (2×10mL). The combined dichloromethane extract was concentrated under reducedpressure and the residue was purified with reverse-phase prep-HPLC toafford the 153 as a pale yellow solid (35 mg, 21%). MS-ESI: [M+H]⁺586.3. ¹H NMR (500 MHz, DMSO-d₆) δ 9.39 (s, 1H), 8.63 (d, J=5.0 Hz, 1H),8.54 (d, J=2.0 Hz 1H), 7.93 (s, 1H), 7.90 (d, J=2.0 Hz, 1H), 7.77 (dd,J=1.5, 12.5 Hz, 1H), 7.60-7.59 (m, 1H), 6.03 (s, 1H), 4.82-4.81 (m, 1H),4.51-4.45 (m, 2H), 4.01-3.98 (m, 2H), 3.77 (s, 3H), 3.62-3.60 (m, 2H),2.90-2.86 (m, 2H), 2.42-2.40 (m, 3H), 1.34 (s, 9H).

Example 154a 1-Methyl-1H-1,2,3-triazol-5-amine 154a

A 50-mL round-bottomed flask was purged with nitrogen and charged with1-methyl-5-nitro-1H-1,2,3-triazole, from Example 149a (0.78 g, 6.09mmol), 10% palladium on carbon (50% wet, 160 mg), and methanol (20 mL).The flask was evacuated, charged with hydrogen gas, and stirred for 4 hat room temperature. The hydrogen was then evacuated and nitrogen wascharged into the flask. The catalyst was removed by filtration through apad of CELITE® and the filtrate was concentrated under reduced pressureto afford 154a (418 mg, 70%) as a yellow solid. MS: [M+H]⁺ 99.3

Example 154b5-Bromo-1-methyl-3-(1-methyl-1H-1,2,3-triazol-5-ylamino)pyridin-2(1H)-one154b

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (20 mL),154a (500 mg, 5.10 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (1362mg, 5.10 mmol), and cesium carbonate (3.325 g, 10.2 mmol). Afterbubbling nitrogen through the suspension for 20 minutes, Xantphos (0.59g, 1.02 mmol) and tris(dibenzylideneacetone)dipalladium(0) (467 mg, 0.51mmol) were added. The system was subjected to three cycles ofvacuum/nitrogen flush and heated at reflux for 5 h. It was then cooledto room temperature and filtered. The filtrate was evaporated underreduced pressure. The residue was purified by silica-gel columnchromatography eluting with 50:1 dichloromethane/methanol to afford 154b(220 mg, 15%) as a brown solid. LCMS: [M+H]⁺ 284.1

Example 154c(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(1-methyl-1H-1,2,3-triazol-5-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 154c

A 25-mL round-bottomed flask equipped with a reflux condenser wascharged with 1 Mb (100 mg, 0.35 mmol),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicacid 116c (174 mg, 0.42 mmol), Pd(dppf)Cl₂ (29 mg, 0.035 mmol), sodiumacetate (57 mg, 0.70 mmol,), K₃PO₄ trihydrate (186 mg, 0.70 mmol), water(6 drops), and acetonitrile (6 mL). After three cycles of vacuum/argonflush, the mixture was heated at 100° C. for 2 h. It was then filteredand the filtrate was evaporated under reduced pressure. The residue waspurified by silica-gel column chromatography eluting with 25:1dichloromethane/methanol to afford 154c (85 mg, 42%) as a brown solid.MS-ESI: [M+H]⁺ 573.4

Example 1546-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[(3-methyltriazol-4-yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one154

A mixture of 154c (85 mg, 0.15 mmol) and lithium hydroxide (36 mg, 1.5mmol) in i-propanol/THF (1:1, 4 mL) and water (1 mL) was stirred at 30°C. for 1 h. The mixture was evaporated under reduced pressure and theresidue was extracted diluted with water (5 mL). It was then with ethylacetate (2×10 mL). The combined ethyl acetate extract was concentratedunder reduced pressure and the residue was purified by reverse-phaseprep-HPLC to afford the title compound (11 mg, 16%) as a white solid.MS-ESI: [M+H]⁺ 531.4. ¹H NMR (500 MHz, DMSO-d₆) δ 8.54-8.53 (m, 2H),8.03 (s, 1H), 7.91 (d, J=1.5 Hz, 1H), 7.80-7.76 (m, 1H), 7.75 (s, 1H),7.55 (d, J=2.5 Hz, 1H), 7.52 (d, J=5.0 Hz, 1H), 6.89 (d, J=2.5 Hz, 1H),5.04 (t, J=5.0 Hz, 1H), 4.34 (d, J=5.0 Hz, 2H), 3.88 (s, 3H), 3.63 (s,3H), 1.40 (s, 9H).

Example 155a2-Nitro-5-(oxetan-3-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine 155a

A mixture of 2-nitro-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine 124d (238mg, 1.40 mmol), oxetan-3-one (252 mg, 3.5 mmol), NaBH₃CN (260 mg, 4.2mmol), and zinc chloride (567 mg, 4.2 mmol) in methanol (10 mL) wasstirred at 50° C. for 6 hours. The mixture was added to water andextracted with dichloromethane three times. The combined organic layerwas concentrated under reduced pressure and the residue was purified bysilica-gel column chromatography eluting with 50:1dichloromethane/methanol to afford 155a (200 mg, 64%). MS: [M+H]⁺ 225.3

Example 155b5-(Oxetan-3-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-amine 155b

A 50-mL single-neck round-bottomed flask was purged with nitrogen andcharged with 155a (0.20 g, 0.89 mmol), 10% palladium on carbon (50% wet,89 mg), and methanol (10 mL). The mixture was evacuated, charged withhydrogen gas, and stirred for 2 h at room temperature. The hydrogen wasthen evacuated and nitrogen was charged into the bottle. The catalystwas removed by filtration through a pad of CELITE® and the filtrate wasconcentrated under reduced pressure to afford 155b (140 mg, 81%). MS:[M+H]⁺ 195.1

Example 155c6-Chloro-2-methyl-4-(5-(oxetan-3-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)pyridazin-3(2H)-one 155c

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (25 mL),155b (640 mg, 3.3 mmol), 4-bromo-6-chloro-2-methylpyridazin-3(2H)-one(1.1 g, 4.95 mmol), Pd₂(dba)₃ (302 mg, 0.33 mmol), XantPhos (381 mg,0.66 mmol), and cesium carbonate (2.15 g, 6.6 mmol). After three cyclesof vacuum/argon flush, the mixture was heated at 100° C. for 3 h. Afterthis time the reaction was cooled to room temperature. It was thenfiltered and the filtrate was evaporated under reduced pressure. Theresidue was washed with ethyl acetate to afford 155c (754 mg, 68%) as ayellow solid. MS-ESI: [M+H]⁺ 337.3

Example 155d(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-(oxetan-3-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-ylamino)-6-oxo-1,6-dihydropyridazin-3-yl)pyridin-3-yl)methylAcetate 155d

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 155c (367 mg, 1.1 mmol),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridine-4-ylboronicacid 116c (908.6 mg, 2.2 mmol), Pd₂(dba)₃ (100.6 mg, 0.11 mmol), P(cy)₃(122.8 mg, 0.44 mmol), Cs₂CO₃ (717 mg, 2.2 mmol), dioxane (20 mL), andwater (0.5 mL). After three cycles of vacuum/argon flush, the mixturewas heated at 110° C. for 2 h. It was then filtered and the filtrate wasevaporated under reduced pressure. The residue was purified bysilica-gel column chromatography eluting with 35:1dichloromethane/methanol to afford 155d (120 mg, 16%) as a yellow solid.MS-ESI: [M+H]⁺ 669.8

Example 1556-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[[5-(oxetan-3-yl)-6,7-dihydro-4H-pyrazolo[1,5-a]pyrazin-2-yl]amino]-6-oxo-pyridazin-3-yl]-2-pyridyl]phthalazin-1-one155

A mixture of 155d (117 mg, 0.175 mmol) and lithium hydroxide.water (73.5mg, 1.75 mmol) in i-propanol/THF (1:1, 8 mL) and water (1 mL) wasstirred at 35° C. for 0.5 h. The mixture was evaporated under reducedpressure and the residue was diluted with water (6 mL). It was thenextracted with dichloromethane (3×50 mL). The combined dichloromethaneextract was concentrated under reduced pressure and the residue waspurified by reverse-phase prep-HPLC to afford 155 (30 mg, 27%) as ayellow solid. MS-ESI: [M+H]⁺ 628.3. ¹H NMR (500 MHz, DMSO-d₆) δ 9.40 (s,1H), 8.64 (d, J=5.5 Hz, 1H), 8.55 (d, J=2.0 Hz, 1H), 7.94 (s, 1H), 7.90(d, J=1.5 Hz, 1H), 7.79-7.77 (m, 1H), 7.61 (d, J=4.5 Hz, 1H), 6.04 (s,1H), 7.61-7.60 (m, 1H), 4.61-4.59 (m, 2H), 4.50-4.45 (m, 4H), 4.01-3.99(m, 2H), 3.78 (s, 3H), 3.69-3.65 (m, 1H), 3.54 (s, 2H), 2.79-2.76 (m,2H), 1.39 (s, 9H).

Example 156a 5-Fluoro-N,N-bis(4-methoxybenzyl)pyridin-2-amine 156

A 100-mL round-bottomed flask was charged with 5-fluoropyridin-2-amine(1.12 g, 10.0 mmol), NaH (288 mg, 12.0 mmol), and THF (20 mL) at 25° C.4-Methoxybenzyl chloride (1.87 g, 12.0 mmol) was added and stirred at25° C. for 2 h. It was then concentrated under reduced pressure. Water(30 mL) was added to the residue and the resulting mixture was extractedwith dichloromethane (3×80 mL). The combined extract was dried overNa₂SO₄, filtered, and evaporated under reduced pressure. The residue waspurified by silica-gel column chromatography eluting with 1:9 ethylacetate/petroleum ether to afford 156a (620 mg, 18%) as a yellow liquid.MS-ESI: [M+H]⁺ 353.0

Example 156b2-(6-(bis(4-Methoxybenzyl)amino)pyridin-3-yl)-2-methylpropanenitrile156b

A 25-mL sealed tube equipped with a magnetic stirrer was charged with156a (528 mg, 1.5 mmol), KHMDS (15 mmol, 15 mL, 1 mol/L of THF), andisobutyronitrile (1.03 g, 15 mmol). After three cycles of vacuum/argonflush, the mixture was heated at 85° C. for 8 h. It was then cooled toroom temperature and quenched with water was added. The mixture wasconcentrated under reduced pressure and the residue was extracted withethyl acetate (3×20 mL). The combined extract was dried over Na₂SO₄,filtered, and evaporated under reduced pressure to afford 156b (514 mg,85%) as a yellow liquid. MS-ESI: [M+H]⁺ 402.0

Example 156c 2-(6-Aminopyridin-3-yl)-2-methylpropanenitrile 156c

A solution of 156b (514 mg, 1.28 mmol) in CF₃COOH (15 mL) was stirred at60° C. for 2 h. After this time the reaction was cooled to roomtemperature. It was then evaporated under reduced pressure and residuewas washed with petroleum ether and ethyl acetate to afford 156c (600mg, crude) as a yellow solid. MS-ESI: [M+H]⁺ 162.3

Example 156d2-(6-(5-Bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yl)-2-methylpropanenitrile156d

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (35 mL),156c (483 mg, 3.0 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (1.6 g,6.0 mmol), Pd₂(dba)₃ (274.5 mg, 0.30 mmol), XantPhos (346.8 mg, 0.60mmol), and cesium carbonate (4.59 g, 15 mmol). After three cycles ofvacuum/argon flush, the mixture was heated at 100° C. for 3 h. Afterthis time the reaction was cooled to room temperature and filtered. Thefiltrate was evaporated under reduced pressure. The residue was purifiedby silica-gel column chromatography eluting with 1:1 ethylacetate/petroleum ether to afford 156d (400 mg, 38%) as a yellow solid.MS-ESI: [M+H]⁺ 347.0

Example 156e(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(5-(2-cyanopropan-2-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 156e

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 156d (346 mg, 1.0 mmol),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicacid 116c (1.65 g, 4.0 mmol), Pd(dppf)Cl₂ (82.5 mg, 0.10 mmol),potassium acetate (196 mg, 2.0 mmol), K₃PO₄ (424 mg, 2.0 mmol),acetonitrile (15 mL), and water (0.5 mL). After three cycles ofvacuum/argon flush, the mixture was heated at 95° C. for 2 h. It wasthen filtered and the filtrate was evaporated under reduced pressure.The residue was purified by silica-gel column chromatography elutingwith 10:1 ethyl acetate/petroleum ether to afford 156e (210 mg, 33%) asa yellow solid. MS-ESI: [M+H]⁺ 635.8

Example 1562-[6-[[5-[2-(6-tert-butyl-8-fluoro-1-oxo-phthalazin-2-yl)-3-(hydroxymethyl)-4-pyridyl]-1-methyl-2-oxo-3-pyridyl]amino]-3-pyridyl]-2-methyl-propanenitrile156

A mixture of 156e (191 mg, 0.30 mmol) and lithium hydroxide.1water (126mg, 3.0 mmol) in i-propanol/THF (1:1, 8 mL) and water (2 mL) was stirredat 35° C. for 0.5 h. The mixture was evaporated under reduced pressureand diluted with water (10 mL). It was then extracted withdichloromethane (3×20 mL). The combined dichloromethane extract wasconcentrated under reduced pressure and the residue was purified byreverse-phase prep-HPLC to afford 156 (30 mg, 17%) as a pale yellowsolid. MS-ESI: [M+H]⁺ 594.3. ¹H NMR (500 MHz, DMSO-d₆) δ 8.83 (s, 1H),8.77 (d, J=2.0 Hz, 1H), 8.59 (d, J=5.0 Hz, 1H), 8.54 (d, J=2.5 Hz, 1H),8.34 (d, J=2.5 Hz, 1H), 7.91 (s, 1H), 7.79-7.74 (m, 2H), 7.57-7.53 (m,2H), 7.40 (d, J=9.0 Hz, 1H), 4.92 (bs, 1H), 4.43 (s, 2H), 3.62 (s, 3H),1.67 (s, 6H), 1.39 (s, 9H).

Example 157a(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-methylisothiazol-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 157a

A 25-mL sealed tube was charged with(4-(5-bromo-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-3-yl)methylacetate 142c (155 mg, 0.28 mmol),4-fluoro-2-(1-oxo-5-methylisothiazol-3-amine hydrochloride (55 mg, 0.33mmol), Cs₂CO₃ (183 mg, 0.56 mmol), Pd₂(dba)₃ (27 mg, 0.030 mmol),XantPhos (35 mg, 0.060 mmol), and DMF (10 mL). After three cycles ofvacuum/argon flush, the mixture was heated at 110° C. under microwaveirradiation for 1 hour. It was then cooled to room temperature andevaporated under reduced pressure. The residue was purified bysilica-gel column eluting with 20:1 methylene chloride/methanol toafford 157a as a yellow solid (64 mg, 39%). MS-ESI: [M+H]⁺ 589.2

Example 1576-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[(5-methylisothiazol-3-yl)amino]-6-oxo-3-pyridyl]-2-pyridyl]phthalazin-1-one157

To a solution of 157a (60 mg, 0.10 mmol) in THF/i-propanol/water (4 mL/4mL/1 mL) was added lithium hydroxide (24 mg, 1.0 mmol). The reactionmixture was stirred at room temperature for 0.5 h and concentrated underreduced pressure. The residue was diluted with water (10 mL) andextracted with ethyl acetate (3×15 mL). The combined organic layer wasdried with Na₂SO₄ and concentrated to afford a yellow solid, which waspurified by reverse-phase prep-HPLC to afford 157 as a yellow solid (27mg, 50%). MS-ESI: [M+H]⁺ 546.7. ¹H NMR (500 MHz, DMSO-d₆) δ 9.28 (s,1H), 8.60-8.54 (m, 3H), 7.90 (s, 1H), 7.78 (d, J=16.0 Hz, 1H), 7.55-7.50(m, 2H), 7.04 (s, 1H), 4.89 (t, J=6.5 Hz, 1H), 4.43-4.41 (m, 2H), 3.61(s, 3H), 2.48 (s, 3H), 1.40 (s, 9H).

Example 158a5-Bromo-3-(5-ethylisoxazol-3-ylamino)-1-methylpyridin-2(1H)-one 158a

A 50-mL single-neck round-bottomed flask equipped with a magneticstirred and a reflux condenser was charged with 5-ethylisoxazol-3-amine(250 mg, 2.23 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (893 mg, 3.35mmol), Pd₂(dba)₃ (102 mg, 0.112 mmol), Xantphos (129 mg, 0.223 mmol),Cs₂CO₃ (1.45 g, 4.46 mmol), and dioxane (20 mL). The system wassubjected to three cycles of vacuum/nitrogen flush and heated at 100° C.under N₂ protection for 3 h. Analysis of the reaction mixture by LCMSshowed complete conversion to the desired product. It was then cooled toroom temperature and filtered. The filtrate was concentrated underreduced pressure and the residue was purified by reverse-phase prep-HPLCto afford 158a (300 mg, 45%) as a white solid. MS-ESI: [M+H]⁺ 297.8

Example 158b3-(5-Ethylisoxazol-3-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one158b

A 50-mL round-bottomed flask equipped with a magnetic stirred and areflux condenser was charged with 158a (250 mg, 0.839 mmol), Pin₂B₂ (320mg, 1.26 mmol), Pd₂(dba)₃ (38.4 mg, 0.042 mmol), X-Phos (48.5 mg, 0.0839mmol), potassium acetate (164.4 mg, 1.678 mmol), and dioxane (20 mL).The system was subjected to three cycles of vacuum/nitrogen flush andheated at 70° C. under N₂ protection for 2 h. Analysis of the reactionmixture by LCMS showed complete conversion to the desired product. Itwas then cooled to room temperature and filtered. The filtrate wasconcentrated under reduced pressure. The residue was washed withpetroleum ether to afford 158b (330 mg, crude) as a dark solid, whichwas used in next step without further purification. MS-ESI: [M+H]⁺ 346.0

Example 158c2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(5-ethylisoxazol-3-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde158c

A 50-mL round-bottomed flask equipped with a reflux condenser wascharged with2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (160 mg, 0.44 mmol), 158b (228 mg, 0.66 mmol), Pd(dppf)Cl₂ (18 mg,0.022 mol), K₃PO₄ (187 mg, 0.88 mmol), sodium acetate (72.2 mg, 0.88mmol), acetonitrile (15 mL), and water (5 drops). The system wassubjected to three cycles of vacuum/nitrogen flush and heated at 100° C.under N₂ protection for 1 h. Analysis of the reaction mixture by LCMSshowed complete conversion to the desired product. It was then cooled toroom temperature and filtered. The filtrate was concentrated underreduced pressure and the residue was purified by silica-gel columnchromatography eluting with 40:1 dichloromethane/methanol to afford 158c(210 mg, 88%) as yellow oil. MS-ESI: [M+H]⁺ 543.3

Example 1586-tert-butyl-2-[4-[5-[(5-ethylisoxazol-3-yl)amino]-1-methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one158

To a solution of 158c (190 mg, 0.35 mmol) in dichloromethane (5 mL) andmethanol (5 mL) was added NaBH₄ (26.5 mg, 0.70 mmol). The reactionmixture was stirred at room temperature for 1 h and quenched withaqueous NH₄Cl. It was then concentrated under reduced pressure and theresidue was extracted with dichloromethane. The combined extract waswashed with brine, dried over Na₂SO₄, filtered, and concentrated underreduced pressure. The residue was purified by reverse-phase prep-HPLC toafford 158 (70 mg, 37%) as white solid. MS-ESI: [M+H]⁺ 545.3. ¹H NMR(500 MHz, DMSO-d₆) δ 9.01 (s, 1H), 8.58 (d, J=5.0 Hz, 1H), 8.54 (d,J=2.0 Hz, 1H), 8.01 (d, J=2.0 Hz, 1H), 7.90 (s, 1H), 7.79 (d, J=13 Hz,1H), 7.57 (d, J=2.5 Hz, 1H), 7.51 (d, J=5.0 Hz, 1H), 6.26 (s, 1H), 4.90(s, 1H), 4.44-4.43 (m, 2H), 3.61 (s, 3H), 2.69-2.65 (m, 2H), 1.40 (s,9H), 1.19 (t, J=8.0 Hz, 3H)

Example 159a 2-(Difluoromethyl)-4-nitro-2H-1,2,3-triazole 159a

A 100-mL single-neck round-bottomed flask containing4-nitro-2H-1,2,3-triazole (500 mg, 4.38 mmol, 1.0 eq.), sodium2-chloro-2,2-difluoroacetate (1310 mg, 8.76 mmol, 2.0 eq.), K₂CO₃ (1140mg, 8.76 mmol, 2.0 eq.), and acetonitrile (20 mL) was stirred at refluxfor 5 h. After cooling to room temperature, the reaction mixture wasfiltered and the filtrate was evaporated under reduced pressure. Theresidue was purified by silica-gel column chromatography eluting with4:1 petroleum ether/ethyl acetate to afford 159a (350 mg, 48%) as abrown liquid. ¹H NMR (500 MHz, CDCl₃) δ 8.38 (s, 1H), 7.39 (t, J=57.5Hz, 1H).

Example 159b 2-(Difluoromethyl)-2H-1,2,3-triazol-4-amine 159b

Following the procedure of Example 151e, and starting with 159a (300 mg,1.83 mmol) and 10% palladium on carbon (50% wet, 60 mg) afforded 159b asa yellow liquid (200 mg, 81%). ¹H NMR (500 MHz, CDCl₃) δ 7.24 (s, 1H),7.14 (t, J=58.5 Hz, 1H), 4.08 (brs, 2H).

Example 159c5-Bromo-3-(2-(difluoromethyl)-2H-1,2,3-triazol-4-ylamino)-1-methylpyridin-2(1H)-one159c

Following the procedure of Example 151f, and starting with 159b (170 mg,1.25 mmol, 1.0 eq.) and 3,5-dibromo-1-methylpyridin-2(1H)-one (504 g,1.89 mmol, 1.5 eq.) afforded 159c as a light yellow solid (280 mg, 70%).MS-ESI:[M+H]⁺ 320.1.

Example 159d(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(2-(difluoromethyl)-2H-1,2,3-triazol-4-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 159d

Following the procedure of Example 152c, and starting with 159c (150 mg,0.46 mmol, 1.0 eq.) and3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicacid (116c) (285 mg, 0.69 mmol, 1.5 eq.) afforded 159d as a yellow solid(90 mg, 32%). MS-ESI:[M+H]⁺ 609.3

Example 1596-tert-butyl-2-[4-[5-[[2-(difluoromethyl)triazol-4-yl]amino]-1-methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one159

Following the procedure of Example 152, and starting with 159d (80 mg,0.13 mmol) afforded 159 as a white solid (29 mg, 40%). MS-ESI: [M+H]⁺567.3. ¹H NMR (500 MHz, CDCl₃) δ 8.70 (d, J=5.0 Hz, 1H), 8.36 (d, J=2.0Hz, 1H), 8.15 (d, J=2.0 Hz, 1H), 7.84 (s, 1H), 7.74 (d, J=2.0 Hz, 1H),7.62-7.53 (m, 4H), 7.25 (t, J=59.0 Hz, 1H), 4.50 (s, 2H), 4.09 (t, J=6.5Hz, 1H), 3.75 (s, 3H), 1.45 (s, 9H).

Example 160a 5-Bromo-1-methyl-3-(pyrazin-2-ylamino)pyridin-2(1H)-one160a

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with pyrazin-2-amine (1.0 g,10 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (2.7 g, 10 mmol),Pd₂(dba)₃ (460 mg, 0.50 mmol), XantPhos (600 mg, 1.0 mmol), cesiumcarbonate (6.52 g, 20 mmol), and 1,4-dioxane (150 mL). After threecycles of vacuum/argon flash, the mixture was heated at 100° C. for 2 h.It was then filtered and the filtrate was evaporated in vacuo. Theresidue was purified by recrystallization with methanol to afford 160a(1.3 g, 47%) as a light green solid. MS-ESI: [M+H]⁺ 281.0.

Example 160b(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-6-oxo-5-(pyrazin-2-ylamino)-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 160b

A 50-mL round-bottomed flask equipped with a reflux condenser wascharged with 160a (140 mg, 0.50 mmol),3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicacid 116c (410 mg, 1.0 mmol), Pd(dppf)Cl₂ (25 mg, 0.025 mmol), K₃PO₄(220 mg, 1.0 mmol), sodium acetate trihydrate (136 mg, 1.0 mmol),acetonitrile (15 mL), and water (0.5 mL). The system was evacuated andrefilled with N₂. The reaction mixture was heated at 100° C. for 1 h. Itwas then cooled to room temperature and filtered. The filtrate wasconcentrated under reduced pressure and the resulting residue waspurified by silica-gel column chromatography eluting withdichloromethane/methanol (100:1 to 25:1) to afford 160b (150 mg, 52%) asa yellow solid. MS-ESI: [M+H]⁺ 570.2.

Example 1606-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-6-oxo-5-(pyrazin-2-ylamino)-3-pyridyl]-2-pyridyl]phthalazin-1-one160

A mixture of 160b (120 mg, 0.21 mmol) and lithium hydroxide monohydrate(88 mg, 2.1 mmol) in THF/i-propanol (4:2, 6 mL) and water (2 mL) wasstirred at 30° C. for 1 h. The mixture was evaporated under reducedpressure and the residue was diluted with water (10 mL). It was thenextracted with ethyl acetate (2×20 mL). The combined ethyl acetateextract was concentrated under reduced pressure and the residue waspurified by reverse-phase prep-HPLC to afford 160 (50 mg, 45%) as awhite solid. MS-ESI: [M+H]⁺ 527.8. ¹H NMR (500 MHz, CHCl₃) δ 8.78 (d,J=2.5 Hz, 1H), 8.71 (d, J=5.0 Hz, 1H), 8.36 (d, J=2.0 Hz, 1H), 8.31 (s,1H), 8.19 (s, 1H), 8.14 (s, 1H), 8.05 (d, J=2.0 Hz, 1H), 7.81 (d, J=2.5Hz, 1H), 7.59 (s, 1H), 7.56-7.55 (m, 2H)

Example 161a1-Methyl-3-(5-methylisoxazol-3-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one161a

A 50-mL round bottomed flask equipped with a magnetic stirrer and areflux condenser was charged with5-bromo-1-methyl-3-(5-methylisoxazol-3-ylamino)pyridin-2(1H)-one 129a(330 mg, 1.16 mmol), Pin₂B₂ (442 mg, 1.74 mmol), Pd₂(dba)₃ (53 mg, 0.058mmol), X-Phos (55 mg, 0.116 mmol), potassium acetate (227 mg, 2.32mmol), and dioxane (20 mL). After three cycles of vacuum/N₂ flush, themixture was heated at 70° C. for 2 h. Analysis of the reaction mixtureby LCMS showed complete conversion to the desired product. It was cooledto room temperature and filtered. The filtrate was concentrated underreduced pressure and the residue was washed with petroleum ether toafford 161a (300 mg, 78%) as a yellow solid. MS-ESI: [M+H]⁺ 332.3

Example 161b2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-methylisoxazol-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)nicotinaldehyde161b

A 100-mL round-bottomed flask equipped with a reflux condenser wascharged with2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloronicotinaldehyde103b (390 mg, 1.08 mmol), 161a (537 mg, 1.62 mmol), cesium carbonate(704 mg, 2.16 mmol), 1,4-dioxane (30 mL), and water (3 mL). Afterbubbling nitrogen through the suspension for 10 minutes, Cy₃P (121 mg,0.43 mmol) and Pd₂(dba)₃ (99 mg, 0.11 mmol) were added. The system wassubjected to three cycles of vacuum/nitrogen flush and heated at refluxfor 3 h. It was then cooled to room temperature and filtered. The solidwas washed with dichloromethane (3×20 mL). The combined filtrate wasconcentrated under reduced pressure and the residue was purified bysilica-gel column chromatography eluting with 20:1 ethylacetate/petroleum ether to afford 161b (300 mg, 52%). MS-ESI: [M+H]⁺528.8

Example 161c6-tert-Butyl-8-fluoro-2-(3-(hydroxymethyl)-4-(1-methyl-5-(5-methylisoxazol-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-2-yl)phthalazin-1(2H)-one161c

To a solution of 161b (300 mg, 0.57 mmol) in methanol (10 mL) was addedNaBH₄ (108 mg, 2.85 mmol) at room temperature. After the reaction wasstirred for 6 h, LCMS indicated the reaction was complete. It was thenquenched with water (10 mL) and concentrated under reduced pressure. Theresulting residue was extracted with dichloromethane (3×20 mL). Thecombined organic layer was washed with brine (20 mL), dried over Na₂SO₄,filtered, and concentrated under reduced pressure. The residue solid waspurified by silica-gel column chromatography eluting with petroleumether/ethyl acetate (1:20 to 100% ethyl acetate) to afford 161c (164 mg,54%) as a white solid. MS-ESI: [M+H]⁺ 530.8

Example 161d Dibenzyl(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-methylisoxazol-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylPhosphite 161d

A mixture of 161c (100 mg, 0.19 mmol), dibenzyldiisopropylphosphoramidite (85 mg, 0.25 mmol), 1H-tetrazole (27 mg, 0.38mmol) in acetonitrile was stirred at room temperature for 1 h. LCMSindicated no further reaction. The mixture containing 161d was used inthe next step without further purification. MS-ESI: [M+H]⁺ 775.3.

Example 161e Dibenzyl(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(1-methyl-5-(5-methylisoxazol-3-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylPhosphate 161e)

To a mixture of 161d in acetonitrile was added m-chloroperbenzoic acid(49 mg, 0.28 mmol). The reaction mixture was stirred at room temperaturefor 5 minutes and filtered. The filtrate was concentrated under reducedpressure and the residue was purified by reverse-phase prep-HPLC toafford 161e (15 mg, 10% two steps) as a white solid. MS-ESI: [M+H]⁺791.3

Example 161[2-(6-tert-butyl-8-fluoro-1-oxo-phthalazin-2-yl)-4-[1-methyl-5-[(5-methylisoxazol-3-yl)amino]-6-oxo-3-pyridyl]-3-pyridyl]methyldihydrogen phosphate 161

A mixture of 161e (10 mg, 0.013 mmol) in trifluoroacetic acid (297 mg,2.60 mmol) was stirred at room temperature for 1 h. The mixture wasconcentrated under reduced pressure and the residue was purified byreverse-phase prep-HPLC to afford 161 (6.7 mg, 84%) as a white solid.MS-ESI: [M+H]⁺ 611.3. ¹H NMR (500 MHz, DMSO-d₆) δ 9.00 (s, 1H), 8.61 (m,1H), 8.50 (bs, 1H), 8.02 (s, 1H), 7.87-7.72 (m, 3H), 7.53 (m, 1H), 6.27(s, 1H), 4.61-4.56 (m, 2H), 3.46 (s, 3H), 2.33 (s, 3H), 1.31 (s, 9H).

Example 162a 5,6-Dihydro-4H-pyrrolo[1,2-b]pyrazole-2-carboxylic Acid162a

A 25-mL round-bottomed flask equipped with a reflux condenser wascharged with ethyl 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-2-carboxylate(540 mg, 3.0 mmol), 2N Aqueous sodium hydroxide solution (3.5 mL), and1,4-dioxane (3.0 mL). The system was heated at 65° C. for 2.5 h. It wasthen cooled to room temperature and adjusted the pH to 2-3 withconcentrated HCl. The solid was collected by filtration to afford 162a(260 mg, 57%) as a yellow solid. MS-ESI: [M+H]⁺ 153.3

Example 162b tert-Butyl5,6-Dihydro-4H-pyrrolo[1,2-b]pyrazol-2-ylcarbamate 162b

A 25-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 162a (197.6 mg, 1.3mmol), tert-butanol (5.0 mL), triethylamine (262.6 mg, 2.6 mmol), andDPPA (550 mg, 2.0 mmol). The system was subjected to three cycles ofvacuum/nitrogen flush and heated at 85° C. for 5 h. It was then cooledto room temperature and purified with Combiflash (A: 1% NH₄HCO₃/water,B: acetonitrile) to afford 162b (45 mg, 15.5%) as a yellow solid.MS-ESI: [M+H-56]⁺ 168.3

Example 162c 5,6-Dihydro-4H-pyrrolo[1,2-b]pyrazol-2-amine 162c

To a solution of 162b (45 mg, 0.20 mmol) in dichloromethane (1.5 mL) wasadded trifluoroacetic acid (1 mL) at room temperature. The solution wasstirred for 5 h. It was then concentrated under reduced pressure toafford 162c which was used in the next step without furtherpurification. MS-ESI: [M+H]⁺ 124.3

Example 162d(2-(6-tert-Butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-(5-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)methylAcetate 162d

A 25-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with(4-(5-bromo-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-3-yl)methylacetate 142c (166.2 mg, 0.30 mmol), 162c (24.6 mg, 0.20 mmol), cesiumcarbonate (130 mg, 0.40 mmol), and 1,4-dioxane (4.0 mL). After bubblingnitrogen through the suspension for 10 minutes, Xantphos (23 mg, 0.040mmol) and tris(dibenzylideneacetone)dipalladium(0) (14 mg, 0.020 mmol)were added. The system was subjected to three cycles of vacuum/nitrogenflush and heated at reflux for 2.5 h. It was then cooled to roomtemperature and filtered. The solid was washed with dichloromethane(3×10 mL). The combined organic layer was concentrated under reducedpressure. The residue was purified by silica-gel column chromatographyeluting with dichloromethane/methanol (100:1 to 50:1 to afford 162d (50mg, 42%) as a yellow solid. MS-ESI: [M+H]⁺ 598.3

Example 1626-tert-butyl-2-[4-[5-(5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-2-ylamino)-1-methyl-6-oxo-3-pyridyl]-3-(hydroxymethyl)-2-pyridyl]-8-fluoro-phthalazin-1-one162

To a solution of 162d (50 mg, 0.080 mmol) in THF/i-propanol/water(1/1/0.5 ml) was added lithium hydroxide (19 mg, 0.80 mmol) at roomtemperature. After the reaction was stirred for 2.5 h, LCMS indicatedthe reaction was complete. Then the mixture was poured into water (15mL) and extracted with dichloromethane (3×20 mL). The combined organiclayer was washed with brine (30 mL), dried over Na₂SO₄, filtered, andconcentrated under reduced pressure. The residue solid was purified byreverse-phase prep-HPLC (A: 1% NH₄HCO₃/water, B: acetonitrile) to afford162 (15 mg, 33.3%) as white solid. MS-ESI: [M+H]⁺ 556.3. ¹H NMR (500MHz, CDCl₃) δ 8.62 (d, J=5.0 Hz, 1H), 8.32 (s, 1H), 7.93 (d, J=2.0 Hz,1H), 7.55-7.47 (m, 4H), 7.26 (s, 1H), 5.74 (s, 1H), 4.47 (d, J=4.0 Hz,2H), 4.07 (t, J=2.0 Hz, 2H), 3.70 (s, 3H), 2.87 (t, J=6.5 Hz, 2H),2.55-2.52 (m, 2H), 1.45 (s, 9H).

Example 901 Biochemical Btk Assay

A generalized procedure for a standard biochemical Btk Kinase Assay thatcan be used to test Formula I and II compounds is as follows. A mastermix minus Btk enzyme is prepared containing 1× Cell Signaling kinasebuffer (25 mM Tris-HCl, pH 7.5, 5 mM beta-glycerophosphate, 2 mMdithiothreitol, 0.1 mM Na₃VO₄, 10 mM MgCl₂), 0.5 μM Promega PTKBiotinylated peptide substrate 2, and 0.01% BSA. A master mix plus Btkenzyme is prepared containing 1× Cell Signaling kinase buffer, 0.5 μMPTK Biotinylated peptide substrate 2, 0.01% BSA, and 100 ng/well (0.06mU/well) Btk enzyme. Btk enzyme is prepared as follows: full lengthhuman wildtype Btk (accession number NM-000061) with a C-terminal V5 and6×His tag was subcloned into pFastBac vector for making baculoviruscarrying this epitope-tagged Btk. Generation of baculovirus is donebased on Invitrogen's instructions detailed in its published protocol“Bac-to-Bac Baculovirus Expression Systems” (Cat. Nos. 10359-016 and10608-016). Passage 3 virus is used to infect Sf9 cells to overexpressthe recombinant Btk protein. The Btk protein is then purified tohomogeneity using Ni-NTA column. The purity of the final proteinpreparation is greater than 95% based on the sensitive Sypro-Rubystaining A solution of 200 μM ATP is prepared in water and adjusted topH7.4 with 1N NaOH. A quantity of 1.25 μL of compounds in 5% DMSO istransferred to a 96-well ½ area Costar polystyrene plate. Compounds aretested singly and with an 11-point dose-responsive curve (startingconcentration is 10 μM; 1:2 dilution). A quantity of 18.75 μL of mastermix minus enzyme (as a negative control) and master mix plus enzyme istransferred to appropriate wells in 96-well ½ area costar polystyreneplate. 5 μL of 200 μM ATP is added to that mixture in the 96-well ½ areaCostar polystyrene plate for final ATP concentration of 40 μM. Thereaction is allowed to incubate for 1 hour at room temperature. Thereaction is stopped with Perkin Elmer 1× detection buffer containing 30mM EDTA, 20 nM SA-APC, and 1 nM PT66 Ab. The plate is read usingtime-resolved fluorescence with a Perkin Elmer Envision using excitationfilter 330 nm, emission filter 665 nm, and 2^(nd) emission filter 615nm. IC₅₀ values are subsequently calculated. Alternatively, theLanthascreen assay can be used to evaluate Btk activity throughquantification of its phosphorylated peptide product. The FRET(Fluorescence Resonance Energy Transfer) that occurs between thefluorescein on the peptide product and the terbium on the detectionantibody decreases with the addition of inhibitors of Btk that reducethe phosphorylation of the peptide. In a final reaction volume of 25 uL,Btk (h) (0.1 ng/25 ul reaction) is incubated with 50 mM Hepes pH 7.5, 10mM MgCl₂, 2 mM MnCl₂, 2 mM DTT, 0.2 mM NaVO4, 0.01% BSA, and 0.4 uMfluorescein poly-GAT. The reaction is initiated by the addition of ATPto 25 uM (Km of ATP). After incubation for 60 minutes at roomtemperature, the reaction is stopped by the addition of a finalconcentration of 2 nM Tb-PY20 detection antibody in 60 mM EDTA for 30minutes at room temperature. Detection is determined on a Perkin ElmerEnvision with 340 nM excitation and emission at 495 nm and 520 nm.Exemplary Btk inhibition IC50 values are in Tables 1, 2, and 3.

Example 902 Ramos Cell Btk Assay

Another generalized procedure for a standard cellular Btk Kinase Assaythat can be used to test Formula I and II compounds is as follows. Ramoscells are incubated at a density of 0.5×10⁷ cells/ml in the presence oftest compound for 1 hr at 37° C. Cells are then stimulated by incubatingwith 10 μg/ml anti-human IgM F(ab)₂ for 5 minutes at 37° C. Cells arepelleted, lysed, and a protein assay is performed on the cleared lysate.Equal protein amounts of each sample are subject to SDS-PAGE and westernblotting with either anti-phosphoBtk(Tyr223) antibody (Cell SignalingTechnology #3531; Epitomics, cat. #2207-1) or phosphoBtk(Tyr551)antibody (BD Transduction Labs #558034) to assess Btkautophosphorylation or an anti-Btk antibody (BD Transduction Labs#611116) to control for total amounts of Btk in each lysate.

Example 903 B-Cell Proliferation Assay

A generalized procedure for a standard cellular B-cell proliferationassay that can be used to test Formula I and II compounds is as follows.B-cells are purified from spleens of 8-16 week old Balb/c mice using aB-cell isolation kit (Miltenyi Biotech, Cat #130-090-862). Testingcompounds are diluted in 0.25% DMSO and incubated with 2.5×10⁵ purifiedmouse splenic B-cells for 30 min prior to addition of 10 μg/ml of ananti-mouse IgM antibody (Southern Biotechnology Associates Cat #1022-01)in a final volume of 100 μl. Following 24 hr incubation, 1μCi³H-thymidine is added and plates are incubated an additional 36 hrprior to harvest using the manufacturer's protocol for SPA [³H]thymidine uptake assay system (Amersham Biosciences # RPNQ 0130).SPA-bead based fluorescence is counted in a microbeta counter (WallaceTriplex 1450, Perkin Elmer).

Example 904 T Cell Proliferation Assay

A generalized procedure for a standard T cell proliferation assay thatcan be used to test Formula I and II compounds is as follows. T cellsare purified from spleens of 8-16 week old Balb/c mice using a Pan Tcell isolation kit (Miltenyi Biotech, Cat #130-090-861). Testingcompounds are diluted in 0.25% DMSO and incubated with 2.5×10⁵ purifiedmouse splenic T cells in a final volume of 100 μl in flat clear bottomplates precoated for 90 min at 37° C. with 10 μg/ml each of anti-CD3 (BD#553057) and anti-CD28 (BD #553294) antibodies. Following 24 hrincubation, 1 μCi ³H-thymidine is added and plates incubated anadditional 36 hr prior to harvest using the manufacturer's protocol forSPA [³H] thymidine uptake assay system (Amersham Biosciences # RPNQ0130). SPA-bead based fluorescence was counted in a microbeta counter(Wallace Triplex 1450, Perkin Elmer).

Example 905 CD86 Inhibition Assay

A generalized procedure for a standard assay for the inhibition of Bcell activity that can be used to test Formula I and II compounds is asfollows. Total mouse splenocytes are purified from spleens of 8-16 weekold Balb/c mice by red blood cell lysis (BD Pharmingen #555899). Testingcompounds are diluted to 0.5% DMSO and incubated with 1.25×10⁶splenocytes in a final volume of 200 μl in flat clear bottom plates(Falcon 353072) for 60 min at 37° C. Cells are then stimulated with theaddition of 15 μg/ml IgM (Jackson ImmunoResearch 115-006-020), andincubated for 24 hr at 37° C., 5% CO₂. Following the 24 hr incubation,cells are transferred to conical bottom clear 96-well plates andpelleted by centrifugation at 1200×g×5 min. Cells are preblocked byCD16/CD32 (BD Pharmingen #553142), followed by triple staining withCD19-FITC (BD Pharmingen #553785), CD86-PE (BD Pharmingen #553692), and7AAD (BD Pharmingen #51-68981E). Cells are sorted on a BD FACSCaliburand gated on the CD19⁺/7AAD⁻ population. The levels of CD86 surfaceexpression on the gated population is measured versus test compoundconcentration.

Example 906 B-ALL Cell Survival Assay

The following is a procedure for a standard B-ALL (acute lymphoblasticleukemia) cell survival study using an XTT readout to measure the numberof viable cells. This assay can be used to test Formula I and IIcompounds for their ability to inhibit the survival of B-ALL cells inculture. One human B-cell acute lymphoblastic leukemia line that can beused is SUP-B15, a human Pre-B-cell ALL line that is available from theATCC.

SUP-B15 pre-B-ALL cells are plated in multiple 96-well microtiter platesin 100 μl of Iscove's media+20% FBS at a concentration of 5×10⁵cells/ml. Test compounds are then added with a final conc. of 0.4% DMSO.Cells are incubated at 37° C. with 5% CO₂ for up to 3 days. After 3 dayscells are split 1:3 into fresh 96-well plates containing the testcompound and allowed to grow up to an additional 3 days. After each 24 hperiod, 50 ul of an XTT solution is added to one of the replicate96-well plates and absorbance readings are taken at 2, 4 and 20 hoursfollowing manufacturer's directions. The reading taken with an OD forDMSO only treated cells within the linear range of the assay (0.5-1.5)is then taken and the percentage of viable cells in the compound treatedwells are measured versus the DMSO only treated cells.

Example 907 CD69 Whole Blood Assay

Human blood is obtained from healthy volunteers, with the followingrestrictions: 1 week drug-free, non-smokers. Blood (approximately 20 mlsto test 8 compounds) is collected by venipuncture into Vacutainer®(Becton, Dickinson and Co.) tubes with sodium heparin.

Solutions of Formula I and II compounds at 10 mM in DMSO are diluted1:10 in 100% DMSO, then are diluted by three-fold serial dilutions in100% DMSO for a ten point dose-response curve. The compounds are furtherdiluted 1:10 in PBS and then an aliquot of 5.5 μl of each compound isadded in duplicate to a 2 ml 96-well plate; 5.5 μl of 10% DMSO in PBS isadded as control and no-stimulus wells. Human whole blood—HWB (100 μl)is added to each well. After mixing the plates are incubated at 37° C.,5% CO₂, 100% humidity for 30 minutes. Goat F(ab′)2 anti-human IgM (10 μlof a 500 μg/ml solution, 50 μg/ml final) is added to each well (exceptthe no-stimulus wells) with mixing and the plates are incubated for anadditional 20 hours. At the end of the 20 hour incubation, samples areincubated with fluorescent labeled antibodies for 30 minutes, at 37° C.,5% CO₂, 100% humidity. Include induced control, unstained and singlestains for compensation adjustments and initial voltage settings.Samples are then lysed with PharM Lyse™ (BD Biosciences Pharmingen)according to the manufacturer's instructions. Samples are thentransferred to a 96 well plate suitable to be run on the BD BiosciencesHTS 96 well system on the LSRII machine. Data acquired and MeanFluorescence Intensity values were obtained using BD Biosciences DIVASoftware. Results are initially analyzed by FACS analysis software (FlowJo). The IC50 for test compounds is defined as the concentration whichdecreases by 50% the percent positive of CD69 cells that are also CD20positive stimulated by anti-IgM (average of 8 control wells, aftersubtraction of the average of 8 wells for the no-stimulus background).The inhibitory concentration (IC50, IC70, IC90) values are calculated byPrism version 5, using a nonlinear regression curve fit.

Example 908 In Vitro Cell Proliferation Assay

Efficacy of Formula I and II compounds are measured by a cellproliferation assay employing the following protocol (Mendoza et al(2002) Cancer Res. 62:5485-5488). The CellTiter-Glo® Luminescent CellViability Assay, including reagents and protocol are commerciallyavailable (Promega Corp., Madison, Wis., Technical Bulletin TB288). Theassay assesses the ability of compounds to enter cells and inhibit cellproliferation. The assay principle is based on the determination of thenumber of viable cells present by quantitating the ATP present in ahomogenous assay where addition of the Cell-Titer Glo reagent results incell lysis and generation of a luminescent signal through the luciferasereaction. The luminescent signal is proportional to the amount of ATPpresent.

A panel of B-cell lymphoma cell lines (BJAB, SUDHL-4, TMD8, OCI-Ly10,OCI-Ly3, WSU-DLCL2) are plated into 384-well plate in normal growthmedium, and serially diluted BTK inhibitors or DMSO alone were added toeach well. Cell viability is assessed after 96 hour incubation byCellTiter-Glo® (Promega). Data may be presented as Relative cellviability in BTK inhibitor-treated cells relative to DMSO-treatedcontrol cells. Data points are the mean of 4 replicates at each doselevel. Error bars represent SD from the mean.

Procedure: Day 1—Seed Cell Plates (384-well black, clear bottom,microclear, TC plates with lid from Falcon #353962), Harvest cells, Seedcells at 1000 cells per 54 μl per well into 384 well Cell Plates for 3days assay. Cell Culture Medium: RPMI or DMEM high glucose, 10% FetalBovine Serum, 2 mM L-Glutamine, P/S. Incubate 0/N at 37° C., 5% CO2.

Day 2—Add Drug to Cells, Compound Dilution, DMSO Plates (serial 1:2 for9 points), Add 20 μl compounds at 10 mM in the 2nd column of 96 wellplate. Perform serial 1:2 across the plate (10 μl+20 μl 100% DMSO) for atotal of 9 points using Precision. Media Plates 96-well conical bottompolypropylene plates from Nunc (cat.#249946) (1:50 dilution) Add 147 μlof Media into all wells. Transfer 3 μl of DMSO+compound from each wellin the DMSO Plate to each corresponding well on Media Plate usingRapidplate.

Drug Addition to Cells, Cell Plate (1:10 dilution), Add 6 μl ofmedia+compound directly to cells (54 μl of media on the cells already).Incubate 3 days at 37 C, 5% CO2 in an incubator that will not be openedoften.

Day 5—Develop Plates, Thaw Cell Titer Glo Buffer at room temperature.Remove Cell Plates from 37° C. and equilibrate to room temperature. forabout 30 minutes. Add Cell Titer Glo Buffer to Cell Titer Glo Substrate(bottle to bottle). Add 30 μA Cell Titer Glo Reagent (Promega cat.#G7572) to each well of cells. Place on plate shaker for about 30minutes. Read luminescence on Analyst HT Plate Reader (half second perwell).

Cell viability assays and combination assays: Cells were seeded at1000-2000 cells/well in 384-well plates for 16 h. On day two, nineserial 1:2 compound dilutions are made in DMSO in a 96 well plate. Thecompounds are further diluted into growth media using a Rapidplate robot(Zymark Corp., Hopkinton, Mass.). The diluted compounds are then addedto quadruplicate wells in 384-well cell plates and incubated at 37° C.and 5% CO2. After 4 days, relative numbers of viable cells are measuredby luminescence using Cell-Titer Glo (Promega) according to themanufacturer's instructions and read on a Wallac Multilabel Reader(PerkinElmer, Foster City). EC50 values are calculated using Prism® 4.0software (GraphPad, San Diego). Formula I or II compounds, andchemotherapeutic agents are added simultaneously or separated by 4 hours(one before the other) in all assays.

An additional exemplary in vitro cell proliferation assay includes thefollowing steps:

1. An aliquot of 100 μl of cell culture containing about 10⁴ cells inmedium is deposited in each well of a 384-well, opaque-walled plate.

2. Control wells are prepared containing medium and without cells.

3. The compound is added to the experimental wells and incubated for 3-5days.

4. The plates are equilibrated to room temperature for approximately 30minutes.

5. A volume of CellTiter-Glo Reagent equal to the volume of cell culturemedium present in each well is added.

6. The contents are mixed for 2 minutes on an orbital shaker to inducecell lysis.

7. The plate is incubated at room temperature for 10 minutes tostabilize the luminescence signal.

8. Luminescence is recorded and reported in graphs as RLU=relativeluminescence units.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. Accordingly, all suitablemodifications and equivalents may be considered to fall within the scopeof the invention as defined by the claims that follow. The disclosuresof all patent and scientific literature cited herein are expresslyincorporated in their entirety by reference.

We claim:
 1. A method of treating an hematological malignancy in apatient comprising administering a therapeutically effective amount of apharmaceutical composition to the patient wherein the pharmaceuticalcomposition comprises a Formula I compound and a pharmaceuticallyacceptable carrier, glidant, diluent, or excipient, wherein the FormulaI compound has the structure:

or stereoisomers, tautomers, or pharmaceutically acceptable saltsthereof, wherein: X¹ is CR¹ or N; X² is CR² or N; X³ is CR³ or N; whereone or two of X¹, X², and X³ are N; R¹, R² and R³ are independentlyselected from H, F, Cl, CN, —CH₃, —CH₂CH₃, —CH₂OH, —CH₂F, —CHF₂, —CF₃,—CH₂CH₂OH, —NH₂, —NHCH₃, —N(CH₃)₂, —OH, —OCH₃, —OCH₂CH₃, and —OCH₂CH₂OH;R⁴ is selected from H, F, Cl, CN, —CH₂OH, —CH(CH₃)OH, —C(CH₃)₂OH,—CH(CF₃)OH, —CH₂F, —CHF₂, —CH₂CHF₂, —CF₃, —C(O)NH₂, —C(O)NHCH₃,—C(O)N(CH₃)₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHC(O)CH₃, —OH, —OCH₃, —OCH₂CH₃,—OCH₂CH₂OH, —OP(O)(OH)₂, cyclopropyl, cyclopropylmethyl,1-hydroxycyclopropyl, imidazolyl, pyrazolyl, 3-hydroxy-oxetan-3-yl,oxetan-3-yl, and azetidin-1-yl; R⁵ is selected from —CH₃, —CH₂CH₃,—CH₂OH, —CH₂F, —CHF₂, —CF₃, —CN, and —CH₂CH₂OH; or two R⁵ groups form a3-, 4-, 5-, or 6-membered carbocyclic or heterocyclic ring; or an R⁵group and an R⁷ group form a 3-, 4-, 5-, or 6-membered carbocyclic orheterocyclic ring; n is 0, 1, 2, 3, or 4; R⁶ is selected from H, —CH₃,—CH₂CH₃, —CH₂CH₂OH, —CH₂F, —CHF₂, —CF₃, —NH₂, —NHCH₃, —N(CH₃)₂, —OH,—OCH₃, —OCH₂CH₃, and —OCH₂CH₂OH; R⁷ is selected from H, —CH₃, —S(O)₂CH₃,cyclopropyl, azetidin-3-yl, oxetan-3-yl, and morpholin-4-yl; Z¹ is CR⁸or N, where R⁸ is selected from H, F, Cl, —CH₃, —CH₂CH₃, —CH₂CH₂OH,—NH₂, —NHCH₃, —N(CH₃)₂, —OH, —OCH₃, —OCH₂CH₃, and —OCH₂CH₂OH; Z² is CR⁹or N, where R⁹ is selected from H, —CH₃, —CH₂CH₃, and —CH₂CH₂OH; and Y¹and Y² are independently selected from CH and N, where Y¹ and Y² are noteach N.
 2. The method of claim 1 wherein the Formula I compound isselected from Formulas Ia-If having the structures:


3. The method of claim 1 wherein X¹ is N, X² is CR², and X³ is CR³. 4.The method of claim 1 wherein X¹ is CR¹, X² is N, and X³ is CR³.
 5. Themethod of claim 1 wherein X¹ is CR¹, X² is CR², and X³ is N.
 6. Themethod of claim 5 wherein X¹ and X³ are CH.
 7. The method of claim 1selected from: X¹ and X³ are N, X¹ and X² are N, or X² and X³ are N. 8.The method of claim 1 wherein X² is CR², and R² is F.
 9. The method ofclaim 1 wherein R⁴ is —CH₂OH.
 10. The method of claim 1 wherein R⁵ is—CH₃, and n is 1 or
 2. 11. The method of claim 1 wherein R⁷ isoxetan-3-yl.
 12. The method of claim 1 wherein Y¹ is CH.
 13. The methodof claim 1 wherein Y² is CH.
 14. The method of claim 1 wherein Z¹ is CH.15. The method of claim 1 wherein Z² is CH.
 16. The method of claim 1wherein the Formula I compound is selected from:6-tert-butyl-8-fluoro-2-(3-(hydroxymethyl)-4-(1-methyl-5-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-2-yl)phthalazin-1(2H)-one;(S)-6-tert-butyl-8-fluoro-2-(3-(hydroxymethyl)-4-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-2-yl)phthalazin-1(2H)-one;(R)-6-tert-butyl-8-fluoro-2-(3-(hydroxymethyl)-4-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-2-yl)phthalazin-1(2H)-one;(S)-6-tert-butyl-8-fluoro-2-(4-(hydroxymethyl)-5-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)pyridin-3-yl)phthalazin-1(2H)-one;6-tert-butyl-2-(4-(5-(5-((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-3-(hydroxymethyl)pyridin-2-yl)-8-fluorophthalazin-1(2H)-one;and(S)-6-tert-butyl-2-(4-(5-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-3-(hydroxymethyl)pyridin-2-yl)-8-fluorophthalazin-1(2H)-one.17. The method of claim 1 wherein the Formula I compound is selectedfrom6-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[1-methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-6-oxo-pyridazin-3-yl]-2-pyridyl]phthalazin-1-one;and6-tert-butyl-8-fluoro-2-[3-(hydroxymethyl)-4-[5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-6-oxo-1H-pyridin-3-yl]-2-pyridyl]phthalazin-1-one.18. The method of claim 1 wherein the hematological malignancy isselected from lymphomas, lymphocytic leukemias, and myeloma.
 19. Themethod of claim 18 wherein the lymphocytic leukemia is Acutelymphoblastic leukemia (ALL), Acute myelogenous leukemia (AML), Chroniclymphocytic leukemia (CLL), Chronic myelogenous leukemia (CML), or Acutemonocytic leukemia (AMOL).
 20. The method of claim 18 wherein thelymphoma is non-Hodgkin lymphoma.
 21. The method of claim 18 furthercomprising administering an additional therapeutic agent selected froman anti-inflammatory agent, an immunomodulatory agent, chemotherapeuticagent, an apoptosis-enhancer, a neurotropic factor, an agent fortreating cardiovascular disease, an agent for treating liver disease, ananti-viral agent, an agent for treating blood disorders, an agent fortreating diabetes, and an agent for treating immunodeficiency disorders.